Final Report Summary - CANCERALIA (Development of novel diagnostic and therapeutic approaches to improve patient outcome in lung and pancreatic tumours) Executive Summary:Epithelial cancers represent 85% of human cancers. Among them, non-small cell lung cancer and pancreatic ductal adenocarcinoma are major causes of death from cancer in the Western world; current projections point to these tumors being the two top causes of cancer death in the US and the EU by 2030 unless there are dramatic therapeutic improvements. These tumors are characterized by primary resistance to common drugs used in cancer therapy and by acquired resistance . Although there is significant heterogeneity in the genomic/genetic landscape of these tumors, certain biochemical features are common to most of them, including the involvement of signalling molecules such as the small GPTases and the biochemical pathways involved in membrane lipid biosynthesis (ie. Kennedy pathway). The long-term objective of the Canceralia project is to increase the survival of patients with lung and pancreatic cancer through the improved understanding of the contribution of these pathways to cancer risk, prognosis, and therapeutic response and through the development of novel therapeutic strategies.Canceralia brings together a coordinated multidisciplinary effort to tackle these issues. The consortium includes molecular oncologists, biochemists, pharmacologists, epidemiologists, and medical oncologists, together with a pharmaceutical industry. The aim is to translate cellular/molecular knowledge into better tools for patient management. These tools should have a direct impact on improving the quality of life and the survival of patients through the development of better strategies for patient stratification, application of novel therapies, and risk identification. Because of the high public health relevance of these two tumors, this should impact on health economy in Europe at multiple levels.Overall, the consortium has succeeded in:- Establishing tools and methods for standardized collection of tumor and normal tissue samples, clinical information, and strategies for sharing these data across laboratories and research groups.- Identifying gene expression signatures characteristic of different tumor subgroups that could be employed for objective patient stratification. - Identifying novel lipidomics profiles distinguishing tumor subgroups robustly that could be employed for patient stratification and for tumor monitoring in response to therapies targeting lipid biosynthetic pathways.- Analyzing prognostic molecular markers of patient outcome in relationship to molecular markers.- Developing tools allowing the correlation of gene expression and lipidomics profiles identified not only using extract-based but also tissue-based techniques. - Establishing patient cohorts for the analysis of genetic factors (variation) associated with cancer risk, patient outcome, and response to therapy.- Establishing biobanks available for future studies of lung and pancreatic cancer with excellent clinical anotation.- Developing in vitro methods for the prediction of response to a major target in the Kennedy pathway for which drugs are in clinical trial. We have demonstrated the existence of different predictors for lung cancer and for pancreatic cancer.- Identifying synergy between drugs targeting the lipid biosynthesis pathway and standard chemotherapies agains lung and pancreatic cancer.- Developing new methods for the synthesis of clinical-grade labeled choline for improved pharmacological and imaging studies in cancer patients.- Identifying novel mechanisms of intrinsic and acquired resistance to choline kinase inhibitors and the molecular pathways involved therein.- Establishing novel targets for future drug development based on the biochemical pathways of interest that can be subject to intellectual property.- Developing and consolidating inter-laboratory collaborations, including short stays of young investigators in different laboratories. This will foster their individual careers and has also strengthened scientific interactions.Project Context and Objectives:Lung and pancreatic cancer are two of the most challenging human tumors. Tobacco smoking is associated with an increased risk of both tumors but the mortality related to them continues to increase (specially in women, for lung cancer). Current projections estimate that these two tumors will be the main causes of cancer death in the next decades unless there are substantial progress in their management. Despite their mortality, lung and pancreatic cancer differ significantly in the challenges posed: early lung cancer is a curable disease but the survival rates for early pancreatic cancer remain very low, in the range of 25% at 5 years. Altogether, there is a dramatic need to improve our understanding of the molecular basis of these tumors because research carried out in the last few years has demonstrated that molecularly-based therapy can impact significantly on tumor treatment and patient survival. This research has also emphasized that: 1) therapies targeting genetic tumor changes can generally induced short-term responses that are followed by the emergence of resistance clones and 2) under each tumor tissue-based designation, a variety of molecular subtypes is hidden, each of them with specific therapeutic sensitivities. These findings underline the concept that therapies targeting generic cellular mechanisms may provide more alternative strategies possibly associated wih less rapid emergence of resistant clones.There is extensive evidence that signalling by small GTPases of the RAS families play numerous and fundamental roles in cancer development/progression through a wide variety of mechanisms. These range from point mutations leading to constitutive activation to protein activation through regulated signalling cascades. Often, both converge, as is the case of Rho proteins for which recent evidence supports a role in cancer through genomic alterations as well as regulated signalling. There is also increasing evidence that common effector pathways (i.e. protein synthesis and cell growth) are particularly interesting because diverse upstream genetic changes converge on their activation (i.e. mTOR, c-MYC, protein biosynthesis, etc.). While mechanisms involved in the regulation of protein synthesis and cell growth have attracted considerable interest, less work has been performed on the molecular pathways that control membrane lipid biosynthesis. This is intriguing given that cells cannot grow and divide without activation of these processes. The Kennedy pathway is crucial for the biosynthesis of membrane lipids and choline kinase is a central enzyme involved therein. Work carried out by members of the consortium, in collaboration with other researchers, has shown that choline kinase inhibition represents a potentially targetable route for cancer treatment in lung, bladder, and colon tumors. The Canceralia consortium was created in this context with the aim of exploiting the potential of these pathways as putative cancer targets both regarding outcome assessment and therapy. In the present project, the notion that the pathway might also impact at the genetic level was raised. Because resistance is proving to be one of the most daunting challenged with targeted therapies, we incorporated these studies into the project so as to be ahead of the hurdles.The Canceralia consortium got together top scientists from a variety of disciplines in order to fulfill its aims: molecular oncologists, biochemists, pharmacologists, epidemiologists, and medical oncologists. It also built on the prior experience (and resources) in lung and pancreatic cancer of the research groups involved but also attracted to these areas investigators who had no prior substantial experience therein. The prior experience of the consortium members allowed the definition of a set of genes (the Canceralia geneset) in which to focus efforts.The main questions tackled by the consortium were:1) What are the changes in Canceralia geneset associated with tumor development?2) Do these changes allow to categorize tumors in subcategories? Can they be recognized in vitro and in vivo?3) Do the Canceralia geneset profiles associate with patient's clinical characteristics and/or outcome?4) Does genetic variation in these genes contribute to cancer risk?5) What are the predictors of response to choline kinase inhibitors?6) What are the main mechanisms of resistance to choline kinase inhibitors?7) How does the activity of choline kinase inhibitors interact with the activity of standard chemotherapy?8) How can the pathway be exploited for improved in vivo diagnosis and pharmacology?9) Can new drug targets be identified through the strategies described above?Project Results:Summary of the main results/foreground of CANCERALIAThe long-term objective of the Canceralia project is to reduce the mortality due to lung cancer and pancreatic cancer, two tumor types for which the current projections are of becoming the top "cancer killers" in the western world by 2030. Such a challenging aim can only be achieved through incremental progress in multiple areas that impact on the risk of developing the disease, the improved taxonomical classification of tumors, the better selection of treatment based on tumor/host features, the development of better therapies (on their own or in combination with current drugs), and the eviction of resistance to therapies which is turning out to be one of the main limitations in the search toward patient's cure. All these aims are, individually, tackled by the Canceralia consortium which was built upon a multidisciplinary expertise of a mix of groups that, in part, had already a long track record of experience working together but also included new partners that could deliver improved interaction and synergy. The consortium included molecular oncologists with experience in cell/molecular cancer biology, lipid biochemists, molecular pharmacologists, imaging specialists (pharma), genetic epidemiologists, and medical oncologists. Eight groups from 7 institutions from 4 EU countries formed the consortium.In WP1, the groups that had access to patients or patient samples (P1, P2, P4, P5) needed to harmonize the approval of procedures by ethical commitees, share informed consents and reach a consensus on the suitability of them for the purposes of the project. This was done within the first months. Additional work in this area was needed during the second report period, specially with regards to WP3 and WP4 (see below). Standard operating procedures for sample collection were also shared and adapted to the individual group settings. Procedures for DNA, and specially for RNA, isolation were shared and a comparison of selected procedures between laboratories was performed in order to ensure that the previous work performed by consortium partners was comparable to the work carried out through the new activities. Both procedures for RNA extraction from fresh frozen and formalin-fixed paraffin-embedded tissues were compared. Collection of clinical information, required for the analysis of risk, prognosis, and response to therapy, was harmonized through the development by P1 of a web-based questionaire/database accessible from any of the study sites and including adequate procedures for quality control of information. This work profited from the existence of ongoing studies by P1 at the European level (in which P2 and P5 participated). Material transfer agreements among the consortium partners were established.In WP2, P4 gathered a large number of NSCLC cases (n=600) for whom annotated biological samples and clinical information were available and RNA was isolated from normal and tumor tissue. After quality control, 400 samples were selected and analyzed by P1. The Canceralia geneset was tested on a training set of 160 cases. The comparison of normal vs. tumor yielded a robust signature distinguishing normal from tumor samples as well as distinct profiles associated with SCC vs. AD features based on gene expression. Genes whose expression was independently associated with outcome were identified, a score was generated and the score was optimized for each tumor stage and reduced for validation strategies. P1 and P4 have selected a total of 16 (plus normalization and control) genes for the validation study which has been performed and results are being generated. A subset of these cases were used for lipidomics studied by P6 who demonstrated the existence of tumor-specific profiles including changes in sphingomyelines, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine. Comparison of normal vs. tumor revealed a dramatic shift towards fatty acid elongation, more prominent in SCC, that could be confirmed through tissue-section based 2D-MS imaging. These changes hold promise for clinical application. The analysis of PDAC has been more challenging due to has difficulties to obtain sufficient tissue from these patients given the fact that this tumor shows a lower incidende and only 20% of patients undergo a resection. To overcome these limitations, P1 has used a collection of patient-derived xenografts in whom transcriptomic analysis has revealed changes in gene expression associated with PDAC (78 Canceralia genes showing up- or down-regulation in tumor vs normal tissue) that could have diagnostic use. Outcome analysis is not possible in this reduced dataset. Lipidomics analysis of the paired samples (n=31) as well of a set of independent patient samples reveals remarkable differences between xenografts and primary tumors.In WP3 the aim was to relate gene expression patterns to response to conventional treatments. This aim has only been partially achieved because of the ethical and methodological constraints derived from the changes in medical treatment of both NSCLC and PDAC during the last 5 years. P1, P4, and P5 are analyzing a set of SNCLC tumors for this purpose but the small sample size of the biopsies obtained from patients with advanced disease, and the need of these samples of molecular-based treatment selection, present a remarkable limitation to these studies.In WP4 the aim was to identify the role of genetic variation in the Canceralia geneset in relationship to risk and prognosis. We compiled genetic variation information on the geneset. Strategies for discovery and replication phases were designed for both NSCLC and PDAC, adapted to the nature of the patient series, samples, and assays available. The association study in both tumors suggests a limited contribution of these pathways to the risk of development of NSCLC and PDAC based on the discovery phase. A candidate gene, DGKK, has been identified holding multiple risk SNPs in LD. The results of the validation phase are being generated and should help to replicate the discovery findings. A very large set of biological samples/data will result from the project (for NSLCL, >800 patients; for PDAC >1500 cases and >500 controls) that will contribute to future studies. The outcome studies will be performed once genotyping has been completed.WP5 and WP6 focus on experimental work aiming at determining the role of ChoKa, a major enzyme in the Kennedy pathway, in the treatment of NSCLC and PDAC. First, P1 identified predictors of resistance to ChoKa-Inh using primary cultures and cell lines. In lung cancer, ASAH1 was identified as a major predictor of resistance; in PDAC, high levels of ChoKa were found to be highly predicitive of response. Genetic and pharmacological strategies were used to confirm these observations in vitro and, in part, in vivo. Immunohistochemical assays carried out by P1, P2, and P3 optimized ChoKa detection for clinical application and establishment of prognosis with positive findings in PDAC. Permanent cell lines with acquired resistance were generated and used for pharmacological studies (see WP7). These results will be crucial to guide clinical trials with ChoKa-Inh.WP7, led by P3 and P7, aimed at developing tools for in vivo diagnosis and molecular pharmacology studies. P3 and P7 have optimized methods for production of labeled choline ready for clinical application in PET. P3 has worked closely with P1 to dissect the molecular mechanisms involved in resistance to ChoKa-Inh in PDAC and has identified a new metabolic process by which choline subproducts are extruded from the cells through ATP-dependent membrane pumps. This appears a more general process relevant to understanding uptake, resistance, and the mechanisms of action of ChoKa-Inh for improved clinical application.In WP8, several of the consortium partners have identified new potential targets for drug development in the lipid biosynthesis pathway. Most notably, P6 and P4 have identified ELOVL6 as a major enzymes involved in fatty acid elongation in NSCLC with in vitro and in vivo validation of activity of inhibitors; P1 has also shown that this enzyme is up-regulated in PDAC. P1 and P6 are working to produce antibodies of adequate quality for clinical application since commercial ones are not specific. Furthermore, P1 has identified the oncogene c-MYC as a major regulator of ELOVL6 and other enzymes in the membrane lipid biosynthesis pathway. The renewed interest in MYC as a drug target emphasizes the relevance of this finding.WP9 refers to the dissemination and exploitation activities. Regarding the former, the fact that the consortium was created de novo and that it generated prospective cohorts of patients results in a delayed output in terms of presentations and publications. Several collaborative papers have already been submitted for publication and many more will be coming out in the next years. There have been numerous presentations at meetings and also to the general population. Finally, several of the results have been deemed suitable for exploitation and due consideration is being given to IP protection.WP10 dealt with project management and Ethical issues.Main results of the CANCERALIA workpackagesThe long-term objective of this proposal is to develop strategies that will increase the response to treatment and survival of patients with two deadly tumors: non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC). To this aim the project proposed to exploit cancer pathways that are important in the majority of human tumors, providing a commonality that is not found with most other targeted therapies. Yet, the project focused on enzymatic driven pathways (lipid biosynthesis) regulated by signalling molecules which in general are more amenable to small molecule drug development. The module of genes/proteins involved in this process has been designated as the "CANCERALIA geneset" (200 genes, focusing on small GTPases and lipid biosynthetic pathways as well as a few literature-selected genes considered to be promising for replication). Work was organized in workpackages covering a wide range of methodological strategies involving multidisciplinary collaboration.WP1. Standards for sample collection, biological material extraction, and data obtention. WP1 involved all the groups implicated in the collection and analysis of samples and patient information. All active Ethics Committee forms were first shared by consortium members to ensure adequacy for the studies proposed within the project. Any changes that had to be introduced were submitted again to Ethics Committees and all studies were finally approved. P1 developed a web-based questionnaire for collection of sociodemographic, clinical, and pathological data that was made available to all partners. This dabase was set to be be linked to molecular and genetic data in order to make available uniform strategies for quality control, data retrieval, and statistical analyses. The questionnaire/database was adapted ad hoc for patients with NSCLC and PDAC. In addition, standardized procedures were established for the isolation of RNA for quantitative analysis of gene expression, adapted both to fresh frozen and formalin-fixed paraffin-embedded tissues, and for DNA extraction. Due to difficulties related to obtaining pancreatic cancer tissue, procedures were adapted for fine needle aspiration samples.WP2. Correlation of the expression levels of Canceralia selected genes with clinical parameters. WP2 focused on the analysis of expression of the Canceralia geneset in tumors and the association of gene expression with clinical parameters used to manage patients in standard clinical care. The main groups involved in these studies were P1 (collection of samples and clinical data and analysis of gene expression), P2 (collection of samples and clinical data), P4 (collection of samples and clinical data), P5 (collection of samples and clinical data), and P6 (lipidomics analysis). A total of 600 non small cell lung cancers (NSCLC) corresponding to patients from whom information and fresh tissue was available was identified. From them, 421 cases were selected for RNA extraction based on a tumor cellularity of >50%; parallel patient-matched normal lung samples were used to extract RNA. These cases were analyzed by P1 and 400 cases were suitable: 160 were included in a discovery screen using the complete Canceralia geneset using TaqMan assays in microfluidic plates . This allowed both global and stage-stratified analyses and the construction of a score for outcome prediction. The analysis showed a robust signature distinguishing normal vs. tumor tissue (17 genes up-regulated and 44 genes down-regulated in tumor samples). In addition, this analysis showed significantly different gene signatures associated with SCC (41 genes) and AD (11 genes) that may also be of clinical interest and that were correlated with lipidomic findings by P6 (see below). Information on expression of 9 genes was used to build a score for tumor recurrence based con Cox multivariable analysis; the predictive value of this score could be achieved with a lower number of 5 genes. The score showed positive association with outcome in all stage groups though the strength was reduced due to lower sample size. Stage-specific optimization was performed for subsequent validation ruling out overfitting of the data. The remaining 240 samples with RNA of adequate quality were used for independent validation using TaqMan assays with a set of 23 promising plus control genes selected for validation. P6 performed lipidomics analysis of NSCLC samples from P4 in two steps, discovery (n=73) and validation (n= 89) (from 206 NSCLC patients provided by P4). A total of 114 and 111 individual phospholipid species significantly discriminated between NSCLC and normal tissue in the discovery and validation set, respectively. Most prominent were a decrease in sphingomyelins (SMs) and an increase in phosphatidylinositols (PIs). In addition, a decrease in multiple phosphatidylserines (PSs) along with an increase in several phosphatidylethanolamines (PEs) and phosphatidylcholines (PCs) were identified. The phospholipid profiles of AD, SCC and matched normal tissue revealed a remarkable shift towards phospholipid species with longer acyl chains in tumors, particularly in SCC. This elongation phenotype was confirmed by 2D-MS using state-of-the art imaging of tissue sections. These changes could significantly be linked with an overexpression of certain fatty acid elongation enzymes identified by P1 in the expression analyses. These studies provide the basis for the potential clinical exploitation of the findings. Phospholipid species could effectively discriminate tumor versus normal tissue and different NSCLC subtypes (AD versus SCC) with an accuracy of 100% and 83.8 %, respectively. However, no significant correlations with clinical outcome could be found. These findings strongly suggest that: 1) multiple, diverse, genetic abnormalities converge on this crucial metabolic pathway and 2) changes in phospholipid profiles occur early during tumor development as a crucial biological component of this process. For PDAC, RNA was isolated from tumor and, when possible, from normal pancreas from the same patient. A minimum content of 50% tumor cellularity was applied. Due to the limited availability of tissue from the resected specimen, normal tissue was not available for all patients. In total, 36 tumor samples and 15 corresponding normal samples were analyzed using global transcriptomics and the Affimetrix HGU 133 plus 2.0 Affimetrix platform. A total of 78 genes were found to be differentially expressed between normal and tissue after applying correction for multiple testing; 51 were overexpressed and 27 underexpressed. As of pancreatic cancer and lipidomics profiles, P1 and P2 have provided P6 with normal-tumor pairs as well as tumor-only samples and 31 patient derived xenografts. The very different cellular composition of normal pancreas and PDAC does not render normal-tumor comparison clinically relevant. The most striking observation is that - unlike in gene expression studies - lipidomics profiles from tumor-derived xenografts display patterns that are very different from those of primary samples (normal and tumor). This is a significant novel finding whose meaning needs to be explored further. However, these observations provide a warning that the complex and abundant stromal composition of PDAC may represent a significant hurdle for the application of lipidomic studies in the clinical setting for this tumor, beyond the difficulties related to obtaining tumor material as discussed in this report.One sub-objective was the exploration of the potential of these findings as markers in the clinical setting. For this, P1, P4, and P6 have analyzed available commercial antibodies that could be of use. Unfortunately, their quality is not acceptable for clinical use (neither for western blotting studies). As per the grant amendment, P1 and P6 have worked on the development of novel monoclonal antibodies against ELOVL proteins and this work is ongoing. Upon their development and characterization, these antibodies could be licensed for commercial application and further analyzed for immunohistochemical analyses.WP3. Role of Canceralia selected genes in treatment response to conventional therapy. The aim of WP3 was to correlate expression levels of the Canceralia geneset with the response to standard treatments in patients with NSCLC and PDAC. In addition, it was proposed to determine phospholipid profiles in this context. The proposal also included to assess these findings in the context of clinical trials. This challeging workpackage has suffered (luckily) from the fact that since submission of the Canceralia grant application there have been radical changes in the management of patients with NSCLC and - more recently - also in PDAC. The former consist of the identification of multiple genetic alterations (first EGFR and KRAS mutations and more recently ALK and ROS fusions) predicting response of tumors to a targeted therapies. In patients with PDAC, Abraxane and FOLFIRINOX have entered the clinical arena. The existence of multiple therapeutic opportunities, but only small biopsies from patients with advanced disease, has rendered obtaining tissue for research studies challenging: 1) in the conventional treatment setting the material is required for molecular diagnosis and 2) in the setting of clinical research sponsored by the industry, this material is scarce and not available to academic researchers. This has not only delayed but also hampered completion of this WP. As indicated in the amendment, P5 has now gathered retrospectively collected frozen material from NSCLC patients that has been sent to P4 for processing so that selected genes of the Canceralia geneset can be tested. However, the small size of the biopsies will not allow to perform lipidomic studies. Of note, the difficulties in completing these tasks reply on an ethical background since part of the available material needs to be preserved in case additional molecular typing needs to be performed. WP4. Genetic variation in relation to susceptibility, prognosis, and treatment response. This WP focused on the identification of genetic variants in the Canceralia geneset that may contribute to the risk of development of lung and pancreatic cancers. The work was based on a two tiered discovery and validation strategy in order to increase the robustness of the findings. In addition, association with outcome has geen explored. First, a review of literature and databases was performed to identify 1500 tag SNPs in 79 "priority" genes. For lung cancer, a retrospective series of NSCLC cases from P1 and P4 was identified. An in-depth association and prognosis analysis was conducted with 41 SNPs in 9 genes with the objective of further selecting interesting SNPs. The associations analyzed did not reach statistical significance on NSCLC risk but SNP associations were found to be significant regarding prognosis and treatment response.The DNA from 530 early stage and 850 advanced stage NSCLC patients from P4 was isolated. A Goldengate technology platform was designed with 1500 SNPs, including those from the discovery phase, and chips were ordered from Illumina. Unfortunately, the chips failed to achieve adequate quality control standards and had to be returned to Illumina. The company, a world leader in this technology, admitted production problems for several projects - including ours - and proposed to switch to the iSelect technology; therefore, new chips were ordered and were to be delivered to P1 in October 2014. We have been extremely disappointed that the new chips again failed to reach the required quality control standards. This is disturbing but it results from unfortunate and unlikely problems that Illumina admits need to be solved. The only effect on the Canceralia project is a further delay but it is beyond the consortium to solve them and to determine a definite date for the solution. We can provide letters from Illumina acknowledging these facts. Unfortunately, we do not think that turning to another company will solve the problem since they are the leaders in the field.Regarding PDAC, P1 and the consortium considered optimal to start with a discovery phase focused on an analysis of information from the PanScan study and then replication, discovery and extension with a new, independent, genome wide analysis. P1 obtained data for >4286 SNPs for 3660 subjects genotyped using lllumina platforms. These SNPs were located in 223 genes belonging to the phospholipid metabolism pathway (Kennedy) and other genes of interest. SNPs were classified according to their position (+/- 20kb). Relevant SNPs were extracted and imputation was performed for missing genotypes dependent upon the study and reference panel allele calls being on the same physical strand of DNA relative to the human genome reference sequence (here, 1000 Genome reference panel). Association with risk was assessed after correction for multiple testing; none of the SNPs survived this stringent filter. Association analyses for the top 20 SNPs in an independent set of 912 cases and 936 controls. After meta-analysis, three SNPs (rs1934188, rs7053160, and rs1934189, in strong LD) in DGKK (diacylglycerol kinase, kappa) survived the multiple testing adjustments (P<1.5*e10-5). DGKK is involved in the conversion of diacylglycerol to phosphatidic acid, an important signalling molecule for growth regulation. Gene.based P-value of association between the Kennedy pathway and pancreatic cancer, based on the gene-set tests, was 0.21. SNP based P-value of association between the Kennedy pathway and pancreatic cancer was 0.195. As of the prospective PDAC study, the opportunity to perform a genomewide screen including the imputation of variants of interest in the Canceralia geneseet was considered exceptionally positive as it broadened the scope of the Canceralia project contribution to a much greater extent than initially planned. Despite the higher cost of this strategy, this has been made possible because P1 had access to the Oncoarray from Illumina (with 500K SNPs) and through additional funding resources that have been incorporated in the project. The PanGen-EU study, led by P1 and contributed by P2 and P5) has recruited 1832 pancreas cancer cases and 1024 controls. Leukocytes/saliva from cases from all participating European countries were sent to Madrid and DNA has been extracted. The genotyping is currently ongoing and is expected to be completed in early 2015; then the statistical analysis will be performed by P1 and we anticipate that this endeavour will have a major impact in the field of PDAC genetic epidemiology. The gathering of clinical data from the patients is also ongoing and will allow to perform an analysis of association with outcome. WP5. Development of a predictive model for the response to treatment with ChoK inhibitors. Based on the strongly developing paradigm of in vitro and in vivo preclinical models predicting response to anticancer drugs, WP1 has used state-of-the art technology to identify markers predictive of response to choline kinase inhibitors (ChoKa-Inh) in NSCLC and PDAC. Tumor tissue from 84 patients with NSCLC undergoing surgical resection was processed by P1 and the sensitivity of primary cultures to the ChoKa-Inh MN58b was assessed. These methods have also been implemented and improved by P4; 33% of the samples tested were highly sensitive whereas 46% were resistant indicating that prediction of response would be of clinical relevance. RNA isolated from MN58b-sensitive and resistant tumors was isolated and expression of Canceralia geneset was assessed by RT-qPCR, leading to the identification of acid ceramidase (ASAH1) as a candidate gene involved in primary resistance. The relevance of this gene was corroborated by the analysis of NSCLC cell lines resistant to ChoKa-Inh also showing up-regulation of ASAH1, which is therefore identified as a candidate marker predictive of response in this tumor. This gene codes for an enzyme involved in the degradation of ceramide into sphingosine and fatty acid that is a candidate for drug development in combination with ChoKa inhibitors.For PDAC, the low resectability rate of this tumor and the marked desmoplastic reaction has not allowed to consistently apply this strategy. However, P1 has studied a panel of PDAC cell lines allowing to determine that levels of ChoK are directly associated with response to ChoKa-Inh in vitro; apoptosis was found as the main mechanism involved in cell growth arrest/death in PDAC cells. Genetic inhibition of ChoKa expression led to increased resistance to pharmacological inhibition, further supporting that ChoKa expression is a predictive marker of therapeutic response. P1, P2, and P3 have optimized immunohistochemical assays of ChoKa in formalin fixed paraffin-embedded clinical tissues and have shown that nuclear - but not cytoplasmic - ChoKa expression is associated with differentiation and survival in patients with PDAC. The finding of nuclear ChoKa in PDAC is concordant with previous studies of P3 and raises issues about the function of this protein in the nucleus. To identify the mechanisms responsible for resistance to ChoKa inhibition, PDAC cell lines with acquired resistance were developed through continued exposure to increasing drug concentrations. Several sensitive-resistant cell pairs were generated but P1 (and P3) focused on IMIM-PC-2 which shows the highest fold-change in IC50 (40-fold). The transcriptomes of parental and resistant cells were compared using RNA-Seq. We did not find evidence of significant modulation of GO pathways is resistant cells. However, single gene comparisons highlighted a remarkable up-regulation of ABCB1 and ABCB4 transporters in the resistant cells. Further protein and functional analysis of these efflux pumps has confirmed the role of drug extrusion in the generation of resistance to MN58b.WP6. Assessment of the response to personalized combinatorial treatments. The main aim of WP6 was to assess the response to combinations of drugs with ChoKa-Inh to develop more personalized therapeutic strategies. This WP was mainly developed by P1 with the contribution of P2 and P3.Regarding NSCLC, P1 focused on the analysis of the combination of cisplatin - the main drug used fro NSCLC - and ChoKa inhibitors. Cultured NSCLC cells treated with variable drug concentrations, either sequentially or concomitantly, showed increased therapeutic efficacy of the combinations in comparison with either drug alone. The identification of ASAH1 as a modulator of resistance in lung cancer also led to in vitro inhibition with drugs targeting this enzyme together with ChoKa-Inh and showed positive results that may have therapeutic impact in clinical trials. Of note, the dramatic changes in the knowledge of the genetic landscape of NSCLC - with profound implications in the management of this tumor - emphasize the need to expand the combinations with therapies targeting oncogenes such as EGFR, ALK or ROS. The large collection of cases gathered by P4 and P5 in this project, for which both gene expression and lipidomics data are available, emphasize the potential for further exploitation of the data gathered together with the molecular analysis of the tumors. Regarding PDAC, at the time of initiation of this grant gemcitabine was considered the standard of care. Therefore, P1 and P2 used combinations of this drug with ChoK inhibitor MN58b to assess their antitumor potential. In addition to testing drug combinations on parental cells, the sensitivity of gemcitabine-resistant cell lines was determined. Resistant cells were had a significantly lower MN58b IC50 than parental cells, indicating potential for the use of ChoKa-Inh in resistant tumors. During the grant period, important studies were published indicating that Abraxane (albumin paclitaxel complex) and FOLFIRINOX (a chemotherapy drug combination containing oxaliplatin and 5-FU) are novel active regimes in patients with PDAC. Therefore, we extended the studies to the combination of ChoKa-Inh with oxaliplatin and 5-FU; in all cases we found at least additive - and in many instances synergistic - effects based on the combination index, supporting the notion that ChoKa inhibition may add to the standard therapeutic options in PDAC. P3 also evaluated choline uptake in response to treatment with the ChoKa-Inh MN58b in all cell lines. Cisplatin treatment was evaluated in BxPC3, MiaPaCa2, and Panc1 cells and gemcitabine treatment was evaluated in Suit2-028 cells. As in NSCLC, the genomic landscape of PDAC has been unraveled in the last few years emphasizing that KRAS is the most common gene involved in this tumor and that new players identified participate mainly in a small fraction of tumors. Therefore, as of now, the main potential for ChoKa inhibition stays either with drugs targeting KRAS and small GTPases or with standard chemotherapy. The in vivo work in PDAC is being completed and results will be available in the first trimester of 2015. WP7. Imaging methods for diagnosis, prognosis, and monitoring. The main goal of WP7 is to develop novel diagnostic and pharmacodynamic strategies for NSCLC and PDAC based on choline kinase activity. P3 and P7 worked to establish methods for the production of alternative precursors to minimise radioactive by-products and improve greater end of synthesis (EOS) radiochemical yields. Two different precursors were synthesised and investigated for 18F-labelling; the unprotected precursor was considered best because it resulted in the fewest radiochemical and chemical by-products, required fewer steps and was of easier preparation. The end product of this task was an automated radiosynthesis of [18F]D4-FCH on a FASTlab platform using disposable cassettes for GMP manufacture suitable for clinical studies. Subsequently, model systems for NSCLC and PDAC were established by P3 to assess the notion that choline kinase levels are critical determinants of [18F]D4-FCH uptake. The pharmacological analysis was performed by P3; first, a panel of 21 NSCLC and SCLC cell lines was screened for ChoKa expression. Three lines (H460, PC9 and A549) were selected for further evaluation as models to represent high, intermediate or low expression of ChoKa. Each chosen cell line shows distinct growth characteristics, paralleled by differing in vivo phenotypes. The dependence of choline uptake on choline kinase activity was tested using MN58b. First, GI50 values were determined for each of the selected cell lines following treatment with MN58b or cisplatin. Next, the time window within which MN58b showed the highest efficacy was established. Growth inhibition was evaluated using the SRB assay with MN58b, cisplatin or dual treatment (MN58b + cisplatin). In parallel, the uptake of the radiolabelled choline analogue [3H]-choline was assessed in H460, PC9 and A549 cells in vitro upon 2-12 hour treatment with MN58b, cisplatin or combination treatment (MN58b + cisplatin). Early changes in uptake of [3H]-choline were correlated to growth inhibition and the value of choline uptake as a predictor of therapeutic response was determined. In vivo, uptake of [18F]D4-FCH in PC9 and H460 xenograft models is ongoing (Task 7.3). Partner 3 is optimising models for this aspect of the work. In vivo assessment of treatment response to vehicle, cisplatin or MN58b was carried out on PC9 lung cancer xenografts and is ongoing in H460 lung cancer xenografts. Treatment regimes were optimized to reduce side effects.Regarding PDAC, in vitro characterisation of choline uptake was evaluated in 5 cell lines, and pairs of cells in which ChoKa knockdown had been established (Suit2 and shCKa-Suit2) or acquired resistance to MN58b was generated (IMIM-PC-2-wt and IMIM-PC-2-R) cell lines. P1, P4, and P3 collaborated on this aspect. Expression of ChoKa was found to be decreased in MN58b-resistant cells. These data allowed the establishment of a working model based on ChoKa expression levels (high and low). In vitro uptake of 3H-choline chloride was found to be consistently associated with ChoKa expression levels across the cell line panel. ChoKa enzymatic activity and conversion of choline to phosphocholine were found to be related to ChoKa expression levels. However, a non-stoichiometric variation between fold change expression, uptake, and ability to phosphorylate suggested that ChoKa may have a role as a regulator of choline uptake independent of its kinase activity. Acquired resistance to MN58b was associated with reduced choline uptake resulting from overexpression of ABCB1 transporter and its inhibition reversed resistance but did not affect activity, suggesting that ChoKa kinase activity, rather than the regulation of cellular transport per se, is the limiting step in intracellular choline retention. Cisplatin was found to variably affect choline uptake in PDAC lines whereas gemcitabine treatment did not. In vivo xenograft experiments showed significant correlation between basal expression levels of ChoKa and growth in Suit2 and IMIM-PC-2 tumours where the silencing or the pharmacologically induced decrease of ChoKa significantly reduced tumour growth. Interestingly, Suit2 tumours and IMIM-PC-2 tumours were found to be very aggressive in mice with early cachexia. P3 has shown that [18F]-D4-Choline uptake was markedly reduced upon genetic of pharmacological inhibition of ChoKa and has demonstrated using HPLC that, in vitro, intracellular [18F]-D4-choline was converted to [18F]-D4-phosphocholine and that large quantities of betaine, betaine aldehyde and phosphocholine were released to the medium. These products are rapidly effluxed out of the cell. This novel mechanism may be more general as it was extended to NSCLC lines. These biochemical findings provide new clues as to the pharmacodynamic assessment of Choka inhibitors in the preclinical and clinical setting. This work is also the basis for ongoing proteomic, phosphoproteomic nad metabolomic analysis cross-correlation analyses.The objective 4 was dropped, after approval by the Commission, due to administrative changes as indicated in the project amendment of 10/10/2013. WP8. Identification of new molecular targets for drug development. WP8 aimed at the identification of novel molecular targets for drug development based on the mechanistic work performed in the prior WP. Four major contributions have been made in the project that provide new molecular targets for combination with ChoKa inhibitors: 1) ASAH1, as a mediator of resistance in NSCLC; 2) ABCB transporters, as mediators of resistance in PDAC; 3) ELOVLs - and in particular ELOVL6 - as proteins involved in the fatty acid elongation phenotype described by P6 in WP2 that might be relevant for cancer therapy; and 4) c-MYC has been identified as a crucial regulator of membrane lipid biosynthesis and ELOVL regulation. The studies related to the first two targets have been outlined earlier.P6 analyzed which of the ELOVLs upregulated in NSCLC cell lines might be contributing to the tumor phenotype. Modulation of ELOVLs in lung SCC cell lines and primary cultures confirmed the involvement of these enzymes in the elongation phenotype. Inhibition of ELOVL6 caused a significant decrease in soft agar colony formation, suggesting a role for acyl chain elongation in cancer growth. Similarly, pharmacological inhibition of this ELOVL in KLN205 tumors in syngeneic DBA/2 mice significantly attenuated lung SCC tumor growth in vivo. Because ELOVL6 might also be of relevance in PDAC, the consortium agreed to place efforts in developing assays to detect ELOVL6 in patient's tumors. A detailed analysis of the specificity of commercial antibodies against ELOVL6 by P4 and P6 showed inadequate quality of these reagents. Therefore, P1 and P6 have set out to develop new monoclonal antibodies that will be screened using western blotting, transfectants, and FFPE tissues. This work is ongoing.A further derivation regarding novel molecular targets comes from the work of P1 who has shown that in PDAC, using a combination of human and mouse models, c-MYC oncogene is a major regulator of the membrane phospholipid synthesis pathway and specifically targets ELOVLs. The direct role of c-MYC in these processes has been shown by genome wide chromatin immunoprecipitation experiments. Given that c-MYC has acquired renewed interest as a therapeutic target in human cancer, our results point to the lipid biosynthesis pathway as an additional readout, and potentially cooperating pathway, in targeting tumors. Additional avenues to exploit drug development have been explored by the consortium. P2 has analyzed the relevance of mutant KRAS to the lipidomic profile of PDAC cells using KRAS knockdown strategies and a combination of clinical samples and cell lines, together with P6. KRAS controls also oxidative vs. non-oxidative metabolism in cancer cells and the levels and activity of G2 cyclins. P5 has shown that miRNAs that are differentially expressed in PDAC patients provide another mechanistic layer that could contribute to the regulation of the Canceralia geneset.WP9. Dissemination and exploitation of results. The development of targeted treatment strategies has emerged as a new fundamental concept in the care of patients with cancer, based on genetic information and personalized approaches. The CANCERALIA project has followed this path to tackle to important and very challenging tumors such as NSCLC and PDAC for which patient outcome is very poor. Over the course of the project it has become clear that a major challenge and an extraordinary opportunity is the increased knowledge on tumor genomics derived from the international or US consortia aimed at dissecting tumor genomes. In addition, this is already translating in novel therapies which render some studies more difficult due to the lack of tumor material available for research studies. Consideration has been given to intellectual property protection. The consortium has submitted several publications, is preparing many additional papers, and has already presented significant parts of this work in scientific meetings. Furthermore, Canceralia has participated in several general community activities (lay).Main exploitable results:- A gene profile associated with NSCLC subclassification.- A gene profile and score associated with stage-specific NSCLC patient survival.- Synthesis of [18F]D4-FCH on FASTlab platform. Initially, the methods will be impletmented for clinical studies. If clinical studies are successful then there is a potential to exploit this method in determination of choline kinase activity in the context of patient management or response assessment.- New targets for drug development either individually or in combination with ChoKa inhibitors.- Under development: production of monoclonal antibodies against ELOVL proteins.WP10. Project management. The Canceralia project was initially led by Dr. J. C. Lacal, a staff member of the CSIC, Spain. After its positive evaluation by the Commission, the CSIC renounced to participate in the project due to the fact that Dr. Lacal and the CSIC itself had IP commitments and financial interests that precluded their participation. This led to Francisco X. Real and the CNIO to take over the coordination of the project. The difficulties encountered at the beginning of the project have rendered the management of the project somewhat more complicated. The willingness of all partners to proceed with the planned work and the good attitude to find solutions have eventually led to an extremely successful project from the scientific standpoint.The Consortium has carried out yearly meetings:- March 2011 (prior to official project initiation) - Madrid, organized by CNIO P1.- May 2012 - Heidelberg, organized by Thoraxklinik Heidelberg P4.- May 2013 - London, organized by ICL P3.- May 2014 - Leuven, organized by U Leuven P6. This meeting was attended by EU officer Dr. Dominika Trzaska who oversaw both the management and scientific progress of the project and discussed relevant difficulties with all partners.In addition, specific project conference calls among partners have been set up in order to discuss the management of the project, specially at the time when there were important decisions to be made. Additional conferences, either with all partners or with a subset of partners, have been held to discuss specific scientific aspects of the project in order to facilitate progress.Three important hurdles that the Consortium had to deal with have been:1) the inability of GE Healthcare P7 to perform the work initially assigned in WP7 due to the relocation of the company from the Hammersmith campus in London. This led initially to the plan that Imanova, Ltd. might take over this part of the project. However, this was not possible. Therefore, an amendment was presented to the Commission reliquishing on objective 4 of WP7 and reassigning resources to other aspects of the project;2) ethical constraints derived from changes in the management of patients - related to advances in genomics - have precluded the completion of WP3. We have firmly believed that all ethical issues take firm priority and have attempted to overcome some of these difficulties but this has also led to incomplete performance in this WP and to delays in performing work. The Coordinator wishes to thank P1, P2, P3 and P4 for their continued efforts to overcomes these difficulties; 3) the genotyping in WP4 was planned to be done using platforms from the world leader in the field, Illumina. However, the company has failed to provide us with appropriate quality controlled chips for the genotyping of NSCLC patient DNA on two occasions. We are convinced, given the expertise of this leading company, that this will be overcome in the next few weeks but this has led to significant delays in completing aspects of WP4. In the case of PDAC, where the consortium opted for using another platform from Illumina, there have been no technical difficulties. This has been an unfortunate problem but we are certain that it will not represent a problem to achieve the final project goals.Management-scientic summary. CANCERALIA represents a coordinated effort by NSCLC and PDAC experts coming from multiple disciplines (molecular oncology, biochemistry, medical oncology, pharmacology, epidemiology, and genetics) to exploit the potential of the lipid synthesis pathways as targets for improved patient management. In addition to underlining the potential of the prime target chosen from the background work (ChoKa), the foreground work performed has identified potential new therapeutic strategies that can be exploited in the drug development, preclinical, and clinical setting .Specifically, we have succeeded in:- Developing a platform for integration of clinical, genetic, epidemiological, and molecular data that can be used across European countries (and languages) for improved data sharing.- Establishing biobanks of patient samples with annotated epidemiological and clinical information that can be used in CANCERALIA as well as in future studies by the EU scientific community.- Increasing the competitiveness of European teams in the area of lipid tumor biology.- The work of a multidisciplinary team has allowed cooperative interaction and the access to patient samples by groups working on fundamental processes in order to obtain detailed lipid profiles associated with tumor phenotypes.- Identifying lipid and genetic profiles associated with lung and pancreatic cancers, highlighint the role of these pathways in these tumors. This work emphasizes that lipid changes likely participate in common pathways whose perturbation is required for tumor development and progression.- Genetic profiling of lung cancers to achieve signatures predictive of tumor progression.- Generating extraordinary resources that - when the work is completed - will have unraveled the role of genetic variation in CANCERALIA geneset in tumor development.- Identifying the in vitro and in vivo efficacy of ChoKa as a therapeutic in NSCLC.- Unraveling several mechanisms of resistance to ChoKa-targeting drugs including some that are known to participate in general in resistance to chemotherapy (i.e. efflux pump) as well as novel ones (i.e. ASAH1).- Identifying novel predictive markers of response to ChKa inhibition such as the levels of expression of this enzyme.- Establishing the synergy between standard chemotherapeutic drugs that are active in NSCLC and PDAC with inhibitors of ChoKa.- Discovering new aspects of choline metabolism related to the effect of ChoKa inhibitors and resistance mechanisms that may be important for the understanding of this therapeutic pathway.- Establishing the molecular basis for personalized treatment of patient's tumors based on molecular profiling.- Developing methods for a more efficient and simple generation of labeled choline to be used in diagnostic and pharmacologic studies in cancer patients (with clinical grade standards of production).- Establishing accurate high throughput biochemical methods for lipid profiling in tissue extracts and extending these studies to the analysis of lipid in tissue sections.- Analyzing the role of KRAS as a major NSCLC and PDAC oncogene in the control of lipid biosynthesis pathway.- Identifying c-MYC as a major regulator of the membrane lipid biosynthesis pathway with the potential therapeutic implications derived thereof.- Providing to the scientific community new therapeutic targets that should contribute to improve the exploitation of ChoKa-in in the clinical setting, both regarding primary treatment and overcoming resistance.Potential Impact:Dissemination and exploitation of results. The development of targeted treatment strategies has emerged as a new fundamental concept in the care of patients with cancer, based on genetic information and personalized approaches. The CANCERALIA project has followed this path to tackle to important and very challenging tumors such as NSCLC and PDAC for which patient outcome is very poor. Over the course of the project it has become clear that a major challenge and an extraordinary opportunity is the increased knowledge on tumor genomics derived from the international or US consortia aimed at dissecting tumor genomes. In addition, this is already translating in novel therapies which render some studies more difficult due to the lack of tumor material available for research studies. Consideration has been given to intellectual property protection. The consortium has submitted several publications, is preparing many additional papers, and has already presented significant parts of this work in scientific meetings. Furthermore, Canceralia has participated in several general community activities (lay).Main exploitable results:- A gene profile associated with NSCLC subclassification.- A gene profile and score associated with stage-specific NSCLC patient survival.- Synthesis of [18F]D4-FCH on FASTlab platform. Initially, the methods will be impletmented for clinical studies. If clinical studies are successful then there is a potential to exploit this method in determination of choline kinase activity in the context of patient management or response assessment.- New targets for drug development either individually or in combination with ChoKa inhibitors.- Under development: production of monoclonal antibodies against ELOVL proteins.The main dissemination activities are:Posters/PresentationsMarien E., Meister M., Muley T., del Pulgar T.G. Lacal J.C. Machiels J., Yinling H., Derua R., Waelkens E. and Swinnen J.V. (2014) Phospholipid profiling of squamous cell carcinoma (SCC) of the lung reveals a marked increase in fatty acyl chain elongation. Keystone Symposium: Tumor Metabolism. 16-21 March. Whistler-Canada.Marien E., Meister M., Muley T., del Pulgar T.G. Lacal J.C. Machiels J., Binda M.M. Van Veldhoven P.P. Yinling H., Derua R., Waelkens E. and Swinnen J.V. (2014) Phospholipid profiling of lung cancer: identification of acyl chain elongation as a novel oncogenic event. Oncoforum. 23 May. KU Leuven-Belgium. (oral presentation) Favicchio R. et al. Preclinical PET/CT evaluation of [18F]-D4-Choline as a diagnostic marker in non-small cell lung cancer, World Molecular Imaging Congress, Savannah 2013.Ferguson R., Neoptolemos J.P. Costello E. and Greenhalf W. Analysis of the effects of K-Ras depletion on the growth characteristics of pancreatic cancer cell lines. Cancer Research UK day, Liverpool 2012 poster.Ferguson R., AbuAlainin W., Neoptolemos J.P. Costello E. and Greenhalf W. Analysis of the effects of K-Ras depletion on the growth characteristics of pancreatic cancer cell lines. National Cancer Research Institute conference, Liverpool 2012, poster.Ferguson R., AbuAlainin W., Neoptolemos J.P. Costello E. and Greenhalf W. Analysis of the effects of K-Ras depletion on the growth characteristics of pancreatic cancer cell lines. Cancer Research UK day, Liverpool 2013 poster.Ferguson R., AbuAlainin W., Ware A., Neoptolemos J.P. Costello E. and Greenhalf W. Analysis of the effects of K-Ras depletion on the growth characteristics of pancreatic cancer cell lines. European Pancreatic Club, Zurich 2013, poster.Ferguson R, Ware A, Bennett L, Armstrong J, AbuAlainin W, Neoptolemos JP, Costello E, Greenhalf W.Analysis of the effects of K-Ras depletion on the growth characteristics of pancreatic cancer cell lines. American Pancreatic Association, Miami 2013, poster. Ferguson R, Ware A, Bennett L, Armstrong J, AbuAlainin W, Neoptolemos JP, Costello E, Greenhalf W. Analysis of the effects of K-Ras depletion on the growth characteristics of pancreatic cancer cell lines. Pancreas Society of Great Britain and Ireland, Liverpool 2013, poster and short presentation based on poster.Ferguson R, AbuAlainin W, Salman A, Neoptolemos JP, Costello E, Greenhalf W. K-Ras regulation of G2 Cyclins via a Cyclin D-independent mechanism. European Pancreatic Club Southampton, 2014 oral presentation. Presentations/SeminarsPresentation of CANCERALIA at the Translational Lung Research Center Heidelberg (TLRC) Faculty Retreat 21-22. July 2014, Schlosshotel Hirschhorn, Hirschhorn, Germany.Marien E., Meister M., Muley T., del Pulgar T.G. Lacal J.C. Machiels J., Derua R., Waelkens E. and Swinnen J.V. (2013) Lipid profiling of squamous cell lung cancer (SCC): identification of changes and applications for diagnostics and therapy. GOA meeting. 13 March. KU Leuven-Belgium. Marien E., Meister M., Muley T., del Pulgar T.G. Lacal J.C. Machiels J., Derua R., Waelkens E. and Swinnen J.V. (2013) Lipid profiling of lung cancer: identification of changes and applications for diagnostics and therapy. LEGENDO seminar. 12 June. KU Leuven-Belgium.Marien E., Meister M., Muley T., del Pulgar T.G. Lacal J.C. Machiels J., Van Veldhoven P.P. Yinling H., Derua R., Waelkens E. and Swinnen J.V. (2014) Phospholipid profiling of lung cancer: identification of acyl chain elongation as a novel carcinogenic event. GOA meeting. 22 January. KU Leuven-Belgium.Marien E., Meister M., Muley T., del Pulgar T.G. Lacal J.C. Machiels J., Binda M.M. Van Veldhoven P.P. Yinling H., Derua R., Waelkens E. and Swinnen J.V. (2014) Phospholipid profiling of lung cancer: identification of acyl chain elongation as a novel carcinogenic event. IUAP meeting. 28 January. Spa-Belgium.Marien E., Meister M., Muley T., del Pulgar T.G. Lacal J.C. Machiels J., Binda M.M. Van Veldhoven P.P. Yinling H., Derua R., Waelkens E. and Swinnen J.V. (2014) Phospholipid profiling of lung cancer: identification of acyl chain elongation as a novel oncogenic event. Oncoseminar at Department Oncology. 28 March. KU Leuven-Belgium.Marien E., Meister M., Muley T., del Pulgar T.G. Lacal J.C. Machiels J., Binda M.M. Van Veldhoven P.P. Yinling H., Derua R., Waelkens E. and Swinnen J.V. (2014) Phospholipid profiling of lung cancer: identification of acyl chain elongation as a novel oncogenic event. Oncoforum. 23 May. KU Leuven-Belgium.Swinnen J. (2013) Lipidomics: Tools and Applications. IUAP, February 2013, Het Pand Ghent, Belgium. Swinnen JV. (2013) Altered phospholipid metabolism in cancer development: applications for cancer diagnosis and treatment. 1st joint Belgian-Netherlands Metabolomics Meeting Spa May 13-14 2013.Swinnen J. (2014) Lipidomics, Cilia and Cancer. Biomedical Student Conference, March 25, 2014, Leuven, Belgium.Swinnen J. (2014) Lipidomics and Cancer; LKI Seminar series, March 25, 2014, Leuven, Belgium.Swinnen J. (2014) Lipid metabolism and Cancer Diagnostics. Imec Academy, September 25, 2014, Leuven, Belgium.Real F. X. (2012) Pathology of the 21st Century – From Molecular Diagnostics to Personalized Cancer Therapy, MDACC Madrid Conference, Madrid, Spain.Real F. X. (2012) Pancreatic cancer: molecular mechanisms in oncogenesis and inflammation. Center for Molecular Medicine, NTNU, Trondheim, Norway.Real F. X. (2014) Pancreatic cancer: molecular mechanisms in oncogenesis and inflammation. Centro Andaluz de Biologia Molecular (CABIMER), Sevilla, Spain.Real F. X. Pancreatic cancer genomics. Roche Workshop on Genomics in clinical practice, Santander, Spain.Real F. X. (2014) What's new in pancreatic cancer 2014 (basic). United European Gastroenterology Federation Meeting, Vienna, Austria.Real F. X. (2014) Pancreatic cancer: molecular mechanisms in oncogenesis and inflammation. Australian Health and Medical Research Meeting, Melbourne, Australia.Carrato A. (2011) New developments in managing pancreatic cancer. ALAGIOS Symposium. Sao Paulo, Brazil.Carrato A. (2013) First line treatment selection impact. Advances in pancreatic cancer. Spanish Society of Medical Oncology Congress. Salamanca, Spain.Carrato A. (2013) Locally advanced pancreatic cancer management. III Bilio-Pancreatic Tumors Symposium. Madrid, Spain.Malats N. (2014) Comorbidities and omics data integration towards a pancreas cancer risk prediction model. Workshop Disease Comorbidities ECCB’14. Strassbourg, France.Malats N. (2014) Pancreas cancer risk: inflammatory-related factors. London Pancreas Workshop. London, UK.Malats N. (2014) Familial Pancreas Cancer, Genetics. Pancreas Cancer Forum, Madrid, Spain.Malats N. (2013) Epidemiology in the "omics" era: will the leaves hide the forest? MDACC Madrid Conference, Madrid, Spain.Malats N. (2013) Epidemiology in the "omics" era: will the leaves hide the forest? Advances in Molecular Oncology: Translating Molecular Biology into Cancer, Sao Paulo School of Advanced Sciences, Sao Paulo, Brasil.Malats N. (2013) The Next Generation of Epidemiological Studies: Insights into Pancreatic and Bladder Cancer. PublicHealth Agency of Canada/Ontario Institute for Cancer Research, Toronto, Canada.Malats N. (2012) The epidemiology of inherited pancreatic disorders. Invited conference, European Pancreatic Club meeting, Prague, Czech Republic.Malats N. (2012) Epidemiology in the "omics" era: will the leaves hide the forest? Invited conference, IDIBELL/ICO, Barcelona, Spain.Malats N. (2012) Epidemiology and omics: will the leaves hide the forest? Centre de Cancerologie de Marseille. Marseille, France.Lectures for lay public:Swinnen J. (2013) Cancer: can we win the war? Brussels Airlines November 28, 2013Swinnen J. (2014) Relay for Life: a researcher’s and personal perspective. Relay for Life Intenational Summit, Sept 27, 2014, Crowne Plaza Hotel Brussels.Swinnen J. (2014) Kanker, kunnen we de strijd winnen? Foundation Against Cancer, Leuven Augustus 27, 2014Swinnen J. (2014) Cancer. Brussels Airlines November 21, 2014, Brussels BelgiumSwinnen J. (2014) Lipid biomarkers in cancer. Science Week for high school students, October 21, 2014, Leuven, BelgiumReal FX. (2012) The Science Week. CNIO, Madrid, SpainReal FX. (2013) The Science Week. CNIO, Madrid, SpainOrganization of workshopsCourse on Pancreatic Pathology "Meet the expert" (co-organizer with N. Malats, I. Esposito, F. Campbell, P. Martinelli), CNIO, Madrid, 2014PublicationsRamírez de Molina A, de la Cueva A, Machado-Pinilla R, Rodriguez-Fanjul V, Gomez del Pulgar T, Cebrian A, Perona R, Lacal JC. Acid ceramidase as a chemotherapeutic target to overcome resistance to the antitumoral effect of choline kinase α inhibition. Curr Cancer Drug Targets. 2012;12:617-24.Mazarico JM, Sanchez-Arevalo Lobo VJ, Favicchio R, Greenhalf W, Costello E, Lacal JC, Aboagye E, Real FX. Choline kinase alpha (CHKA) as a therapeutic target in pancreatic ductal adenocarcinoma: expression, predictive value, and sensitivity to inhibitors. Submitted.Martinelli P, Madriles F, Canamero M, Carrillo-de-Santa Pau E, del Pozo N, Guerra C, Real FX. The acinar regulator GATA6 suppresses KRasG12V-driven pancreatic tumorigenesis in mice. Submitted (second review in Gut).Marien E., Meister M., Muley T., Fieuws S., Bordel S., Derua R., Spraggins J., Van de Plas R., Dehairs J., Wouters J., Bagadi M., Dienemann D., Thomas M., Schnabel P.A. Caprioli R.M. Waelkens E. and Swinnen J.V. (2014) Non-small cell lung cancer (NSCLC) is characterized by dramatic changes in phospholipid profiles. Submitted.Marien E., Meister M., Muley T., del Pulgar T.G. Derua R., Spraggins J., Van de Plas R., Dehairs J., Machiels J., Binda M.M. Brown J.W. Yinling H., Dienemann D., Thomas M., Schnabel P.A. Caprioli R.M. Lacal J.C. Waelkens E. and Swinnen J.V. (2014) Phospholipidomics identifies acyl chain elongation as a novel mediator of tumor growth in non-small cell lung cancer. In preparation.Sanchez-Arevalo Lobo VJ, Moreno U, Mazarico JM, et al. c-Myc oncogene controls a wide membrane lipid biosynthesis programme required for malignant transformation. In preparation. Favicchio F, Brickute D, Fortt R, Twyman F, Lacal JC, Aboagye EO. Choline metabolism is an early predictor of EGFR-induced cell survival in NSCLC. 2015 Manuscript in preparation for submission to Cancer Res.Favicchio R, Brickute D, Mazarico JM, Real FX, Eric O. Aboagye EO. A novel choline metabolite efflux mechanism in pancreatic cancer. 2015 Manuscript in preparation for submission to Cancer Res.List of Websites:www.canceralia.euCoordinator contact details:Francisco X. RealFBBVA-CNIO Cancer Cell Biology ProgrammeSpanish National Cancer Research Center - CNIOMelchor Fernández Almagro, 328029-MadridSpainTel. +34917328000 ext. 3660E-mail: freal@cnio.es
