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Breast cancer; Evidence-generating care; Risk-adjusted prevention Cáncer de mama; Atención generadora de evidencia; Prevención ajustada al riesgo Càncer de mama; Atenció generadora d'evidències; Prevenció ajustada al risc Background: Risk-adjusted cancer screening and prevention is a promising and continuously emerging option for improving cancer prevention. It is driven by increasing knowledge of risk factors and the ability to determine them for individual risk prediction. However, there is a knowledge gap between evidence of increased risk and evidence of the effectiveness and efficiency of clinical preventive interventions based on increased risk. This gap is, in particular, aggravated by the extensive availability of genetic risk factor diagnostics, since the question of appropriate preventive measures immediately arises when an increased risk is identified. However, collecting proof of effective preventive measures, ideally by prospective randomized preventive studies, typically requires very long periods of time, while the knowledge about an increased risk immediately creates a high demand for action. Summary: Therefore, we propose a risk-adjusted prevention concept that is based on the best current evidence making needed and appropriate preventive measures available, and which is constantly evaluated through outcome evaluation, and continuously improved based on these results. We further discuss the structural and procedural requirements as well as legal and socioeconomical aspects relevant for the implementation of this concept. The project was funded by the German Federal Ministry of Health (grant No. 2515FSB401 to Rita Schmutzler and Christiane Woopen) for supporting the international expert meetings, and a grant of the EU Horizon 2020 program, BRIDGES (grant No. 634935, PI Peter Devilee, WP5-PI Rita Schmutzler), for the compilation of the most recent findings of genetic risk prediction.

BackgroundPatients with cancer, especially haematological cancer, are at increased risk for breakthrough COVID-19 infection. However, so far, a predictive biomarker that can assess compromised vaccine-induced anti-SARS-CoV-2 immunity in cancer patients has not been proposed.MethodsHere, we employed machine learning approaches to identify a biomarker signature based on blood cytokine and growth factors linked to vaccine response from 199 cancer patients receiving BNT162b2 vaccine.ResultsWe show that C-reactive protein (CRP; general marker of inflammation), interleukin (IL)-15 (a pro-inflammatory cytokine), IL-18 (interferon-gamma inducing factor), and placental growth factor (an angiogenic cytokine) can correctly classify patients with a diminished vaccine response assessed at day 49 with >80% accuracy. Amongst these, CRP showed the highest predictive value for poor response to vaccine administration. Importantly, this unique signature of vaccine response was present at different studied timepoints both before and after vaccination and was not majorly affected by different anti-cancer treatments.ConclusionWhile we propose a blood-based signature of cytokines and growth factors that can be employed in identifying cancer patients at continued risk of COVID-19, our data also importantly suggest that such a signature could reflect the inherent make-up of some cancer patients who are also refractive to immunotherapy.

BackgroundResidents of nursing homes (NH) are at high risk of COVID-19 related morbidity and death and may respond poorly to vaccination because of old age and frequent comorbidities.MethodsForty residents and forty staff members either naïve or previously infected with SARS-CoV-2 were recruited in two NH in Belgium before immunization with two doses of 30µg BNT162b2 mRNA vaccine at day 0 and day 21. Binding antibodies (Ab) to SARS-CoV-2 receptor binding domain (RBD), spike domains S1 and S2, RBD Ab avidity, and neutralizing Ab against SARS-CoV-2 wild type and B.1.351 variant were assessed at days 0, 21, 28, and 49.ResultsSARS-CoV-2 naïve residents had lower Ab responses to BNT162b2 mRNA vaccination than naïve staff. These poor responses involved lower levels of IgG to all domains of the vaccine antigen, lower avidity of RBD IgG, and lower levels of Ab neutralizing the vaccine strain. No naïve resident had detectable neutralizing Ab to the B.1.351 variant. High and comparable Ab responses were observed in residents and staff previously infected with SARS-CoV-2. Clustering analysis revealed that poor vaccine responders not only included naïve residents but also naïve staff, emphasizing the heterogeneity of responses to mRNA vaccination in the general population.ConclusionsThe poor Ab responses to mRNA vaccination observed in infection naïve residents and in some naïve staff members of NH suggest suboptimal protection against breakthrough infection, especially with variants of concern. Adapted vaccination regimens may be needed to provide optimal protection against COVID-19 to vulnerable populations.SummaryPoor antibody responses to COVID-19 mRNA vaccination were observed in SARS-CoV-2 infection naïve residents and some naïve staff members of nursing homes. This suggests suboptimal protection against breakthrough infection, especially with variants of concern, and the need for adapted vaccination regimens.

Plants are constantly exposed to changes in their environment which require permanent adjustment of their cellular metabolism. Transgenic plants harboring chimeric fusions between 5'-regulatory sequences of stress genes and a reporter gene, together with immunochemical analyses, documented tissue and organelle specificity of the respective gene products. Approaches using transgenic plants which harbored chimeric gene fusions between 5'-regulatory promotor sequences of stress proteins linked to selectable markers or reporter genes, in combination with random or directed mutagenesis, will provide an elegant "stress regulatory gene" retrieval system. The transgenic plants harbored high resistance toward larvae of the lepidopteran Manduca sexta, at toxin concentrations similar to the ones routinely applied by spraying commercial dry-powder Bt toxin. Indeed, cotransforming a pea lectin gene and CpTI generated transgenic plants with increased resistance compared to plants which harbored only either of both genes. Transgenic plants expressing viral CP, were found to be highly protected toward viral infection.

BACKGROUND Patients with pancreatic cancer do not feel involved in the development of their treatment and care plans. In Belgium, these plans are decided on during multidisciplinary team meetings. However, limited time is spent on the discussion of the preferences of the patient during these meetings. This research project aims to develop a patient-reported experience measure (PREM) for pancreatic cancer and assess if its use can support collaborative treatment decision-making. OBJECTIVE This paper aims to outline the protocol for a multi-method research project to improve person-centered pancreatic cancer care in Belgium. Three subobjectives are pursued: (1) to develop a PREM to assess the experiences of care-related aspects in pancreatic cancer care, (2) to validate the PREM, and (3) to develop and evaluate an educational intervention to support the use of the PREM’s results. METHODS For the development of the PREM, an exploratory mixed methods study design will be used. The study will start with a survey followed by a telephone interview involving patients with pancreatic cancer and digestive oncology health care professionals. Study two is the testing of the content and construct validity of the PREM. Study three involves the implementation study according to the Medical Research Council framework of a complex intervention introducing the PREM in practice. The effectiveness of the intervention will be investigated using a pragmatic randomized controlled trial study design. RESULTS The protocol presents the entire structure of the research project. Ethics approval to conduct the exploratory mixed methods study (objective 1) has been obtained, and recruitment has started since January 2022. CONCLUSIONS The poor prognosis of patients with pancreatic cancer should not be considered a hurdle to not study this patient population group. Involving patients in the research and decision-making processes early on is key. This project aims to realize a scientifically sound research process providing research outputs that can easily and timely be implemented in the care trajectory of patients with pancreatic cancer. This research project will also lead to recommendations on how to involve patients with pancreatic cancer and how the methodology of this research project can be translated to other patient groups. INTERNATIONAL REGISTERED REPORT PRR1-10.2196/29004

Background: Cancer patients are at higher risk of developing severe COVID-19. However, safety and efficacy of COVID-19 vaccination in cancer patients undergoing treatment is unclear. Methods: In this interventional prospective multi-cohort study, priming and booster doses of the BNT162b2 COVID-19 vaccine were administered 21 days apart to solid tumor patients receiving chemotherapy, immunotherapy, targeted- or hormonal therapy, and patients with a hematologic malignancy receiving rituximab or after allogeneic hematopoietic stem cell transplantation. Primary endpoint was anti-SARS-CoV-2 receptor binding domain (RBD) antibody level at 28 days post-booster. Anti-RBD IgG titers above 200 IU/ml was used to define high-responders as it leads to a neutralization response required for 50% protection against symptomatic SARS-CoV-2 infection in 268 independent subjects. Secondary endpoints included self-reported adverse events (AEs), antibody response over time and number of breakthrough infections after vaccination. Findings: 28 days post-booster vaccination of 197 cancer patients, antibody response was lower in the hematological cohort (geometric mean titer (GMT) 17·61 IU/mL [95% CI 7·17-43·24]) and the chemotherapy cohort (GMT 234·05 IU/mL [95% 95%CI 122·10-448·66]) when compared to healthy individuals (n=40; GMT 1844·93 IU/mL [95% CI 1383·57-2460·14]), irrespective of timing of vaccination during chemotherapy cycles. Only 55% and 7% of patients receiving chemotherapy or rituximab could be categorized as high-responders at 28 days post-booster. During the study period, five subjects tested positive for SARS-CoV-2 infection, including three after full vaccination. Local and systemic AEs were mostly mild to moderate, with only 1-3% of patients experiencing severe AEs. Local, but not systemic, AEs occurred more frequently after booster than after priming dose. Interpretation: The BNT162b2 vaccine is well-tolerated in cancer patients under active treatment. However, the antibody response of immunized cancer patients was delayed and diminished, mainly in patients receiving chemotherapy or rituximab, resulting in breakthrough infections. Trial Registration: EudraCT number 2021-000300-38 Funding: The regulatory sponsor was the Antwerp University Hospital (EudraCT number 2021-000300-38). The trial was funded by the Belgian Government through Sciensano (COVID-19_SC004, COVID- 19_SC059, COVID-19_SC061). Declaration of Interest: MP declares to have an advisory role within Remedus, all other authors declare no competing interests. Ethical Approval: The study was approved by the local ethics committee and was executed in accordance with Good Clinical Practice and the Declaration of Helsinki (ICH GCP E6(R2)).

Next-generation sequencing (NGS) is being integrated into routine clinical practice in the field of (hemato-) oncology to search for variants with diagnostic, prognostic, or therapeutic value at potentially low allelic frequencies. The complex sequencing workflows used require careful validation and continuous quality control. Participation in external quality assessments (EQA) helps laboratories evaluate their performance and guarantee the validity of tests results with the ultimate goal of ensuring high-quality patient care. Here, we describe three benchmarking trials performed during the period 2017&ndash, 2018 aiming firstly at establishing the state-of-the-art and secondly setting up a NGS-specific EQA program at the national level in the field of clinical (hemato-) oncology in Belgium. DNA samples derived from cell line mixes and artificially mutated cell lines, designed to carry variants of clinical relevance occurring in solid tumors, hematological malignancies, and BRCA1/BRCA2 genes, were sent to Belgian human genetics, anatomic pathology, and clinical biology laboratories, to be processed following routine practices, together with surveys covering technical aspects of the NGS workflows. Despite the wide variety of platforms and workflows currently applied in routine clinical practice, performance was satisfactory, since participating laboratories identified the targeted variants with success rates ranging between 93.06% and 97.63% depending on the benchmark, and few false negative or repeatability issues were identified. However, variant reporting and interpretation varied, underlining the need for further standardization. Our approach showcases the feasibility of developing and implementing EQA for routine clinical practice in the field of (hemato-) oncology, while highlighting the challenges faced.

Background Cancer control has seen very rapid advances over the past couple of decades. Introduction of vaccines, launch of organised population-based screening programmes as well as a consistently enhanced survival and cure after treatment for care. Unfortunately, this has not been matched with an adequate harmonised and balanced approach at the health policy level. European Union launched Joint Actions (JAs) as an instrument to support joint European definition of key issues and their solutions through the work under a partnership approach in identifying good national practices in cancer control as well as proposing development of policies in previously poorly managed or neglected areas, such as survivorship and rehabilitation. Some of the instruments, such as guidelines for screening have not always been up-to-date to meet the advances brought by adjacent fields (such as HPV vaccination in prevention of cervical cancer) or challenges proposed by the broader professional communities. Results Literature and systematic reviews, setting definitions for several important areas, such as comprehensive cancer control networks or survivorship, agreement on quality criteria and defining standards for screening programmes, for comprehensive cancer control networks and addressing socio-economic inequalities in cancer control have all resulted from the activities of the joint actions. The first JA EPAAC developed a panorama of directions for several dimensions of cancer control and provided policymakers with the European Guide on National Cancer Plans. The second JA CanCon proposed a joint critical review of the three recommended screening programmes and took a position on the four new proposed ones (prostate, lung, ovary and stomach). It also carried out an extensive systematic literature review on the problem of the socio-economic differences and inequalities in cancer care and control. For the first time, a policy paper dealing with public health genomics was developed. Similarly, the complex issue of rational reallocation of resources in cancer care was also tackled with. Conclusions Consistent collaboration at the multinational level through joint financing by both - the European Union as well as its Member States - seems to be providing good outcomes and brings cancer control to the top of the policy agenda. By using this mechanism, it was possible to identify issues in cancer policy that had been neglected, such as survivorship, psychosocial rehabilitation, socio-economic inequalities and palliative care. At the same time, instruments were developed, which should serve as tools for health policymakers in advancing their level of quality in cancer policy in the future. Partnership collaboration between the European Commission and its Member States was fruitful in providing innovative approaches and solutions to some of the most important challenges that cancer policy and cancer control are facing. Key messages Joint work on policy recommendations between European Commission and Member States was fruitful. Joint Actions provide up-to-date recommendations on important challenges in cancer control.

Human genomics is undergoing a step change from being a predominantly research-driven activity to one driven through health care as many countries in Europe now have nascent precision medicine programmes. To maximize the value of the genomic data generated, these data will need to be shared between institutions and across countries. In recognition of this challenge, 21 European countries recently signed a declaration to transnationally share data on at least 1 million human genomes by 2022. In this Roadmap, we identify the challenges of data sharing across borders and demonstrate that European research infrastructures are well-positioned to support the rapid implementation of widespread genomic data access.

The Table 1 (EU declaration signatory and membership status) of the original article published online contained several errors: the IARC was incorrectly listed in the table, which only includes countries, and footnote; Croatia, Lithuania, Poland and Portugal were not listed as full EMBL members; Estonia is now listed as a Prospect Member of EMBL; Hungary, Ireland and Israel were incorrectly listed as BBMRI-ERIC members; Iceland was omitted from the table. Hyphens stand for ‘not applicable’; the table and footnote have been corrected accordingly. These errors have now been corrected online and in print.

Identification of genetically modified (GM) plants remains a difficult task, especially when few information on the GM content in a sample is available. The CaMV P-35S is a commonly applied genetic element in GM plants and represents as such a suitable starting point for the tracing of GM plants. Here, the application of a CaMV P-35S LT-RADE genome walking method on five different GM crops is documented: MON810 maize, LLRICE62 rice, T45 rapeseed, A2704-12 soybean and LLCOTTON25 cotton. Two sets of oligonucleotide primers are presented as a potential forward and reverse genome walking strategy with genomic DNA as template. The method’s applicability for assessing the recombinant nature of P-35S positive signals is discussed in detail. This study demonstrates the general use of LT-RADE in molecular characterization of GMOs retrieving several previously unknown transgenic insert nucleotide sequences. Current limitations and future developments are discussed.

In order to provide reliable and harmonized information on methods for GMO (genetically modified organism) analysis we have published a database called “GMOMETHODS” that supplies information on PCR assays validated according to the principles and requirements of ISO 5725 and/or the International Union of Pure and Applied Chemistry protocol. In addition, the database contains methods that have been verified by the European Union Reference Laboratory for Genetically Modified Food and Feed in the context of compliance with an European Union legislative act. The web application provides search capabilities to retrieve primers and probes sequence information on the available methods. It further supplies core data required by analytical labs to carry out GM tests and comprises information on the applied reference material and plasmid standards. The GMOMETHODS database currently contains 118 different PCR methods allowing identification of 51 single GM events and 18 taxon-specific genes in a sample. It also provides screening assays for detection of eight different genetic elements commonly used for the development of GMOs. The application is referred to by the Biosafety Clearing House, a global mechanism set up by the Cartagena Protocol on Biosafety to facilitate the exchange of information on Living Modified Organisms. The publication of the GMOMETHODS database can be considered an important step toward worldwide standardization and harmonization in GMO analysis.

The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GMmaize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semiurban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GMpollen, sample DNA extraction and real-time PCR analysis. Our results suggest that this ‘GM pollen monitoring by bioaerosol sampling and PCR screening’ approach might represent an useful aid in the surveillance of GM-free areas, centres of origin and natural reserves., JRC.I.3-Molecular Biology and Genomics

Simple tools to detect the presence of genetically modified organisms (GMO) in commercial products represent a valuable aid in managing the legal requirements for GMO testing in a cost-effective way. The “Real-Time PCR-Based Ready-to-Use Multi-Target Analytical System for GMO Detection” was developed to meet such requirements and was here further tested for its applicability on detecting GMO in recalcitrant maize matrices. Sixty-four processed maize products were purchased from the market of the European Union and analyzed for their GMO content. Seventy-five percent of the test samples were positive for the presence of GMO. In two samples, trace amounts of the so-called asynchronously authorized GMO could be detected. The overall outcome of the analyses indicated that most of the commercially available genetically modified maize events for food use could be detected in this study. Finally, the possibility to use the “Real-Time PCR-Based Ready-to-Use Multi-Target Analytical System for GMO Detection” in detecting GMO at a 0.1% mass level is documented. The implications of these results on the further development of such type of PCR-based GMO detection systems are discussed.

Identification of crops present in food and/or feed matrices represents an important step in the screening strategies targeting genetically modified organisms (GMO). Soybean, maize, oilseed rape, rice, cotton, sugar beet and potato are to date the most important sources of genetically modified materials imported in the European Union (EU). In order to allow detection of their presence in an integrated screening approach, a set of SYBR®Green real-time polymerase chain reaction (qPCR) methods has been developed which can be used under the same assay conditions and at similar efficiency for each of the abovementioned crops. Each qPCR method is shown to meet the performance criteria (i.e. specificity, limit of detection and PCR efficiency) set by the European Network of GMO Laboratories (ENGL). When combined with the equivalent qPCR methods targeting GMO elements, these crop-specific SYBR®Green qPCR methods can aid the development of an efficient tool for determining GMO presence in food and/or feed products.

e18543Background: Cancer pathology is strongly driven by genetic modifications in the genome DNA what invoked the development of integrated technology in assessing cancer preventive and curative st...

The Cauliflower Mosaic Virus “35S promotor” (p35S) and the Agrobacterium “Nopaline Synthase” terminator (tNOS) are the most represented generic recombinant elements in commercial genetically modified crops to date. A set of four new SYBR®Green qPCR methods targeting the “p35S” and “tNOS” core elements have been developed. These qPCR methods generate short amplicons of 147 and 75 bp for the “p35S” element and 172 and 69 bp for the “tNOS” element. Single target plasmids containing these amplicons were constructed and allow determining the nominal melting temperature (Tm value) of each amplicon. The four methods are specific for their respective targets, and moreover, three of them are highly sensitive (up to 1–2 copies detectable) at a PCR efficiency ranging between 95 and 100%. The latter methods can detect their respective targets at 0.1% (w/w) gDNA levels and are suitable for detecting low levels of genetically modified materials containing the “p35S” and/or “tNOS” elements.

The determination of the presence of genetically modified plant material by the detection of expressed genetically engineered proteins using lateral flow protein strip tests has been evaluated in different matrices. The presence of five major genetically engineered proteins (CP4-EPSPS, CryIAb, Cry9C, PAT/pat and PAT/bar protein) was detected at low levels in seeds, seed/leaf powder and leaf tissue from genetically modified soy, maize or oilseed rape. A comparison between “protein strip test” (PST) and “polymerase chain reaction” (PCR) analysis of genetically modified food/feed samples demonstrates complementarities of both techniques.

Scientists have been looking at the role the immune system may play in fighting cancer for decades, but it is only recently that this knowledge has been translated into immuno-oncology therapies that provide the promise of long-term quality survival to patients [1–3]. Agents are now available to patients with advanced melanoma [1] and castration resistant prostate cancer, [3] and many more agents are expected to become available in the next few years, including in some of most common (e.g. breast and lung) and most difficult-to-treat cancers [1–5]. What makes these new therapies unique is that they confer long-lasting memory to the immune system, enabling it to adapt itself to fight against cancer cells even after remission. Side-effects also tend to be manageable compared to many other cancer therapies. But as with all new treatments, providing fast access to immuno-oncology therapies to appropriate patients will be increasingly challenging in our resource-constrained health care systems. In addition, the science of immuno-oncology is rapidly evolving, and we are still learning how to use these agents to achieve the best possible response in patients [4]. We still lack a clear understanding of what biological factors make a given immuno-oncology therapy work in a particular patient [6]. Also, the clinical development pathways traditionally used in oncology were typically developed for conventional cytotoxic therapies and may be poorly suited to reflect the effects seen with immunooncology therapies [7]. With these challenges in mind, the European Expert Group on Immuno-Oncology was established in early 2014 as an independent network of patients, oncology healthcare professionals, scientists, industry and politicians to help improve overall understanding of what immuno-oncology therapies are and what they may mean for cancer patients. A first goal of the Expert Group was to help policymakers identify what policy adaptations were needed to enable rapid access to immunooncology therapies in appropriate patients across Europe. The resulting Policy Action Framework [8] was launched at the European Parliament in November 2014 along with a guide for patients on immuno-oncology [9]. The Action Framework proposes five priority areas, as shown in Figure 1. Figure 1. Key recommendations for policymakers on immuno-oncology [8]. Promoting a greater understanding of immuno-oncology among policymakers, regulatory and Health Technology Assessment (HTA) agencies, health professionals and patients is key. Informed policymakers are more likely to integrate new therapies into cancer plans and policies, and ensure that appropriate resources and funding are put in place to deliver on these plans at the local level. Professional training across the entire care team is essential for treatments to be used as appropriately as possible in patients who are most likely to benefit, and to monitor the long-term effects, including toxicity, of this new group of agents. And patients need continuous information and support in order to engage fully in treatment decisions. Long-term quality survival is the ultimate goal of cancer therapy [10], however obtaining this evidence may take several years. Therefore, it is important to identify validated intermediary endpoints that are predictive of long-term survival [11]. Integrating appropriate measures of quality of life into clinical trials and clinical practice is also critical to assess the impact of new therapies on patients both during and after treatment. Also, HTA agencies and subsequent funding decisions should be based on what matters most to patients and their families, and take account of the full societal benefits of new therapies, for example individuals’ ability to return to work and lead a ‘normal’ life with long-term survival. Greater flexibility is also needed in existing regulatory frameworks to allow for an appropriate balance between the need for safety and efficacy data and rapid access to promising therapies [12]. For example, the European Medicines Agency is currently exploring more flexible frameworks which allow regulatory decisions to be based on an evolving set of data for selected therapies [13, 14] Early collaboration between drug developers, patient organisations, regulatory agencies and HTA agencies is instrumental for the success of such schemes, as they need to agree to a comprehensive development and licensing plan early on, matched with adaptive frameworks for reimbursement and HTA decisions as well. Collaborative, multidisciplinary research platforms in the form of public-private partnerships (PPPs) also have a huge role to play in immuno-oncology. For example, the Innovative Medicines Initiative is Europe’s largest PPP aimed at speeding up the development of, and patient access to, innovative medicines, particularly in areas where there is high unmet patient need [15]. PPPs may also help provide access to immuno-oncology therapies in poorer-resourced countries as part of compassionate use programmes. Collaborations between the research community and pharmaceutical companies may also allow the creation of large databases that combine data from several clinical trials. These data may help identify which patient characteristics make individuals more likely to respond to different immuno-oncology therapies and develop suitable biomarkers and other patient selection tools as a result. Finally, mechanisms need to be put in place to make sure that the most effective new therapies are included in local care pathways and supported by appropriate funding streams, particularly in heavily decentralised health care systems. For example, the Melanoma Taskforce in the UK developed a national toolkit for local commissioners responsible for funding care to ensure equitable, timely and appropriate access to the full range of approved treatments for people with advanced melanoma across all localities. Long-term alternative financing mechanisms that address payer concerns and maximize and accelerate patient access will also be critical.

Combinatory SYBR®Green real-time PCR Screening (CoSYPS) is an efficient, sensitive approach for detecting complex targets such as genetically modified organisms (GMOs) in food and feed products. GMO analysis for legal purposes has become increasingly complex and costly due to the diversity in recombinant targets present in the different GMOs. For this reason, screening for the presence of GMOs is in general the first step in the detection of GM material in a product. CoSYPS allows detecting the large majority of globally commercial GMOs using SYBR®Green real-time PCR methods for six GM targets (P35S, Tnos, CryIAb, CP4-EPSPS, PAT and BAR) combined with species-specific PCR methods (e.g., maize, soy, rapeseed). Here, the results of an inter-laboratory trial on seven samples with different GMO mixtures at different levels are presented. In total, 13 laboratories participated in the trial and the currently most frequently used PCR analysis platforms are represented. The inter-laboratory study clearly demonstrates that PCR methods used in CoSYPS form a very robust GMO screening system. Sensitivity, specificity, positive and negative predictive values are for all PCR methods higher than 95 % for all samples. Together, these results show that the SYBR®Green real-time PCR methods used in CoSYPS are effectively applicable to different PCR platforms and amendable to configuration into a sensitive high-throughput GMO screening and decision support tool.

Background: Since their first commercialization, the diversity of taxa and the genetic composition of transgene sequences in genetically modified plants (GMOs) are constantly increasing. To date, the detection of GMOs and derived products is commonly performed by PCR-based methods targeting specific DNA sequences introduced into the host genome. Information available regarding the GMOs’ molecular characterization is dispersed and not appropriately organized. For this reason, GMO testing is very challenging and requires more complex screening strategies and decision making schemes, demanding in return the use of efficient bioinformatics tools relying on reliable information. Description: The GMOseek matrix was built as a comprehensive, online open-access tabulated database which provides a reliable, comprehensive and user-friendly overview of 328 GMO events and 247 different genetic elements (status: 18/07/2013). The GMOseek matrix is aiming to facilitate GMO detection from plant origin at different phases of the analysis. It assists in selecting the targets for a screening analysis, interpreting the screening results, checking the occurrence of a screening element in a group of selected GMOs, identifying gaps in the available pool of GMO detection methods, and designing a decision tree. The GMOseek matrix is an independent database with effective functionalities in a format facilitating transferability to other platforms. Data were collected from all available sources and experimentally tested where detection methods and certified reference materials (CRMs) were available. Conclusions: The GMOseek matrix is currently a unique and very valuable tool with reliable information on GMOs from plant origin and their present genetic elements that enables further development of appropriate strategies for GMO detection. It is flexible enough to be further updated with new information and integrated in different applications and platforms., JRC.I.3-Molecular Biology and Genomics

In 2010, 184 Genetically Modified (GM – see glossary) events, representing 24 crops have already received worldwide regulatory approval. To date, 29 countries have cultivated GM crops, whereas 59 countries have granted regulatory approvals for their import for food and feed use and release into the environment. The six main countries cultivating GM crops are USA, Brazil, Argentina, India, Canada and China. In the EU the cultivation area of biotech crops amounts only 0,1%of the cultivation area reaching 125 million hectares in 25 countries (Stein & Rodriguez-Cerezo, 2009). The most important biotech crop is soybean (50% of the biotech crops cultivation area), followed by maize (31%), cotton (14%) and oilseed rape (4%) (James, 2010).

Escherichia coli is a group of bacteria which has raised lots of concerns in the recent years. Five major intestinal pathogenic groups have been recognized among which the verocytotoxin or shiga-toxin (stx1 and/or stx2) producing E. coli (VTEC or STEC respectively). Due to the high number of outbreaks related to VTEC strains, the European Food Safety Authority (EFSA) has requested the monitoring of the “top-five” serogroups (O26, O103, O111, O145 and O157) most often encountered in foodborne diseases; it has also addressed the need for validated methods. High Resolution Melting analysis allows the discrimination among very similar sequences, with stx sequences being such an example. Therefore, we developed a real-time PCR flexible system capable of rapidly detecting the presence of the toxin genes together with intimin (eae) in the case of VTEC, or aggregative protein (aggR) in the case of the O104:H4 strain, which was responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature, and they were further multiplexed so that they would demand less time and initial DNA material. Moreover, HRM analysis allowed for the discrimination among strains possessing different profiles, meaning different combination of traits. The development, application to food samples and the possible flexibility of the methods are thoroughly discussed. A decision support system based on the amplification and melting data has also been developed to support the detection., JRC.I.3-Molecular Biology and Genomics

Genetically modified plants, in the following referred to as genetically modified organisms or GMOs, have been commercially grown for almost two decades. In 2010 approximately 10% of the total global crop acreage was planted with GMOs (James, 2011). More than 30 countries have been growing commercial GMOs, and many more have performed field trials. Although the majority of commercial GMOs both in terms of acreage and specific events belong to the four species: soybean, maize, cotton and rapeseed, there are another 20 + species where GMOs are commercialized or in the pipeline for commercialization. The number of GMOs cultivated in field trials or for commercial production has constantly increased during this time period. So have the number of species, the number of countries involved, the diversity of novel (added) genetic elements and the global trade. All of these factors contribute to the increasing complexity of detecting and correctly identifying GMO derived material. Many jurisdictions, including the European Union (EU), legally distinguish between authorized (and therefore legal) and un-authorized (and therefore illegal) GMOs. Information about the developments, field trials, authorizations, cultivation, trade and observations made in the official GMO control laboratories in different countries around the world is often limited, despite several attempts such as the OECD BioTrack for voluntary dissemination of data. This lack of information inevitably makes it challenging to detect and identify GMOs, especially the un-authorized GMOs. The present paper reviews the state of the art technologies and approaches in light of coverage, practicability, sensitivity and limitations. Emphasis is put on exemplifying practical detection of un-authorized GMOs. Although this paper has a European (EU) bias when examples are given, the contents have global relevance., This review has partly been made possible as a result of the EU-funded integrated project Co-Extra http://www.coextra.eu (contract FOOD-2005-CT-007158) and discussions within an ad hoc working group of the European Network of GMO Laboratories (ENGL) over several years. AHJ is grateful for financial support from the Norwegian Research Council (grants 170363 and 178288). MP is grateful for financial support from the Spanish Ministerio de Ciencia e Innovación (project AGL2010-17181).

While many decisions rely on real time quantitative PCR (qPCR) analysis few attempts have hitherto been made to quantify bounds of precision accounting for the various sources of variation involved in the measurement process. Besides influences of more obvious factors such as camera noise and pipetting variation, changing efficiencies within and between reactions affect PCR results to a degree which is not fully recognized. Here, we develop a statistical framework that models measurement error and other sources of variation as they contribute to fluorescence observations during the amplification process and to derived parameter estimates. Evaluation of reproducibility is then based on simulations capable of generating realistic variation patterns. To this end, we start from a relatively simple statistical model for the evolution of efficiency in a single PCR reaction and introduce additional error components, one at a time, to arrive at stochastic data generation capable of simulating the variation patterns witnessed in repeated reactions (technical repeats). Most of the variation in Cq values was adequately captured by the statistical model in terms of foreseen components. To recreate the dispersion of the repeats’ plateau levels while keeping the other aspects of the PCR curves within realistic bounds, additional sources of reagent consumption (side reactions) enter into the model. Once an adequate data generating model is available, simulations can serve to evaluate various aspects of PCR under the assumptions of the model and beyond., JRC.I.3-Molecular Biology and Genomics

European Commission regulation 2073/2005 on the microbiological criteria for food requires that Escherichia coli is monitored as an indicator of hygienic conditions. Since verocytotoxigenic E. coli (VTEC) strains often cause food-borne infections by the consumption of raw food, the Biological Hazards (BIOHAZ) panel of the European Food Safety Authority (EFSA) recommended their monitoring in food as well. In particular, VTEC strains belonging to serogroups such as O26, O103, O111, O145, and O157 are known causative agents of several human outbreaks. Eight real-time PCR methods for the detection of E. coli toxin genes and their variants ( stx 1 , stx 2 ), the intimin gene ( eae ), and five serogroup-specific genes have been proposed by the European Reference Laboratory for VTEC (EURL-VTEC) as a technical specification to the European Normalization Committee (CEN TC275/WG6). Here we applied a “modular approach” to the in-house validation of these PCR methods. The modular approach subdivides an analytical process into separate parts called “modules,” which are independently validated based on method performance criteria for a limited set of critical parameters. For the VTEC real-time PCR module, the following parameters are being assessed: specificity, dynamic range, PCR efficiency, and limit of detection (LOD). This study describes the modular approach for the validation of PCR methods to be used in food microbiology, using single-target plasmids as positive controls and showing their applicability with food matrices.

Today GMO analysis has become an integral part in the development of new genetically modified plants, in subsequent breeding, seed production and verification programmes. GMO analysis is a key technology in export and import of agricultural commodity products, for ascertaining regulatory compliance of GMOs in different countries, for labelling requirements, and for product authenticity and traceability. In Europe, in particular, GMO analysis is implemented in all Member States to fulfil legal requirements regarding GMOs and GMO-derived products, their release into the environment, cultivation, importation and their utilisation as food, food ingredients and animal feed (European Commission 2001, 2003a, 2003b). A key technical element for the authorisation of GMOs within the EU is the provision of an event-specific quantitative detection method validated according to internationally accepted standards to allow the control and monitoring of a particular GMO along the production, processing and the distribution chain. In this context the European Commission Joint Research Centre (JRC) has the mission to provide scientific and technical support to EU policy development on GMO. The nominated European Union Reference Laboratory for GM Food and Feed (EURL-GMFF) at the JRC is working at the forefront of GMO analysis in Europe. Legally responsible for method validation under Regulation (EC) No 1829/2003 (European Commission 2003a), the EURLGMFF works in close collaboration with the European Network of GMO Laboratories not only in method validation but also in the harmonisation of a number of topics spanning from sampling to method development, data analysis and measurement of uncertainty. In addition, in Europe, the EURL-GMFF plays a key role in the official food controls according to Regulation (EC) No 882/2004 (European Commission 2004a). Still, the multi-factorial nature of GMO analysis, the diverse and often complex composition of the samples under examination, the increasing number of GM events and, finally, the

The detection of genetically modified (GM) materials in food and feed products is a complex multi-step analytical process invoking screening, identification, and often quantification of the genetically modified organisms (GMO) present in a sample. "Combinatory qPCR SYBRGreen screening" (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials in products. The CoSYPS decision-support system (DSS) interprets the analytical results of SYBRGREEN qPCR analysis based on four values: the C(t)- and T(m) values and the LOD and LOQ for each method. A theoretical explanation of the different concepts applied in CoSYPS analysis is given (GMO Universe, "Prime number tracing", matrix/combinatory approach) and documented using the RoundUp Ready soy GTS40-3-2 as an example. By applying a limited set of SYBRGREEN qPCR methods and through application of a newly developed "prime number"-based algorithm, the nature of subsets of corresponding GMO in a sample can be determined. Together, these analyses provide guidance for semi-quantitative estimation of GMO presence in a food and feed product.

The steady rate of development and diffusion of genetically modified plants and their increasing diversification of characteristics, genes and genetic control elements poses a challenge in analysis of genetically modified organisms (GMOs). It is expected that in the near future the picture will be even more complex. Traditional approaches, mostly based on the sequential detection of one target at a time, or on a limited multiplexing, allowing only a few targets to be analysed at once, no longer meet the testing requirements. Along with new analytical technologies, new approaches for the detection of GMOs authorized for commercial purposes in various countries have been developed that rely on (1) a smart and accurate strategy for target selection, (2) the use of high-throughput systems or platforms for the detection of multiple targets and (3) algorithms that allow the conversion of analytical results into an indication of the presence of individual GMOs potentially present in an unknown sample. This paper reviews the latest progress made in GMO analysis, taking examples from the most recently developed strategies and tools, and addresses some of the critical aspects related to these approaches.

We have compared the effects of two elicitors of defense-related processes on rice (Oryza sativa L.) suspension cells. Both chitosan and salicylic acid induced the accumulation of extracellular chitinase, thickening of the cell wall, and a variety of cytological changes in treated cells. Chitosan also induced the production of a brown pigment and cell death. Both of these effects depended on the availability of reactive oxygen species, because the damage was greatly reduced by either catalase or free-radical scavengers. Pretreating cells with salicylic acid also protected them from the cytotoxic effects of chitosan. This type of induced tolerance persisted when salicylic acid was removed and was not simply due to the release of extracellular substances, because salicylic acid-treated cells did not protect untreated cells from chitosan-induced death. Salicylic acid also stimulated the production of a 10-kilodalton subtilisin inhibitor that was not produced by chitosan-treated cells. Most of these changes are associated with the hypersensitive response of many plant species, including monocotyledons, and may serve as an in vitro model for investigating the biochemistry of some diseases.