Center For Translational Molecular Medicine Research Articles

Dominant frequency predicts sinus rhythm maintenance after electrical cardioversion for persistent atrial fibrillation in men but not in women

Abstract Background Women are usually underrepresented in studies evaluating rhythm control strategies in patients with atrial fibrillation (AF). Subsequently, the same holds true for studies looking at predictors for success of a rhythm control strategy for AF. Purpose To study the predictive power of the non-invasively determined dominant frequency (DF) on the electrocardiogram (ECG) in men and women undergoing electrical cardioversion (ECV) for persistent AF. Methods We matched 105 female patients undergoing elective ECV for persistent AF and 105 male control patients based on age and cardiovascular comorbidity profile. We determined the DF on all 12 leads of a standard digital 10 seconds ECG recorded on the day of ECV. Recurrences of AF within the first year after ECV were documented. Results There were no differences in comorbidities, AF duration, left ventricular systolic function, indexed left atrial volume and anti-arrhythmic drugs between male and female patients. The dominant frequency was significantly lower in male patients without an AF recurrence on all leads. The best performing lead to identify patients with recurrences was lead III with an AUC 0.752. The optimal cut-off point was a DF <5.98 Hz with a sensitivity 84% and a specificity 67%. There was no significant difference in DF between female patients with and without an AF recurrence. The AUC in lead III was 0.47 (Figure 1). Conclusion The non-invasively measured dominant frequency is able to predict AF recurrence after electrical cardioversion in male patients with persistent AF but not in a matched female cohort. This difference might be explained by different pathophysiological mechanisms underlying AF in male and female patients. Therefore, future research is needed on pathophysiological differences between men and women that can explain and might overcome these challenges. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): European Network for Translational Research in Atrial Fibrillation (grant no. 261057), the Center for Translational Molecular Medicine (COHFAR),

Abstract OT3-07-01: Efficacy of magnetic resonance-guided high intensity focused ultrasound for the ablation of breast cancer

Abstract Background: Over the past decades, breast cancer treatment has evolved from extensive disfiguring surgery to less invasive procedures. In patients with small localized tumors breast conserving therapy (BCT) has become the standard of care. However, surgery still involves risks associated with general anesthesia and complications such as bleeding, infection and suboptimal cosmetic results. Conversely, Magnetic Resonance-guided High intensity Focused Ultrasound (MR-HIFU) ablation of breast cancer is an entirely non-invasive procedure that can be performed under procedural sedation and analgesia (PSA). With MR-HIFU, a focused ultrasound beam penetrates through soft tissues and causes localized heating (55-70°C) of a target. Magnetic resonance imaging (MRI) is used for target-identification and real-time temperature-monitoring (MR thermometry). In a previous phase I study in ten breast cancer patients, tumors were deliberately partially ablated and safety and accuracy of a dedicated MR-HIFU breast system (Profound Medical) was shown.1,2 This system’s lateral sonication approach and wide aperture reduce the risk of heating heart, lungs and skin to a minimum.3 In the current study we aim to demonstrate that complete breast tumor ablation with MR-HIFU is feasible. Trial design: Single-arm phase II study in patients with primary breast cancer. We will use a dedicated MR-HIFU breast system (Profound Medical) to ablate breast tumors before BCT or mastectomy, in a treat-and-resect protocol. We will perform MRI before MR-HIFU, during the MR HIFU session and one week after to evaluate radiologic response. Primary endpoint is efficacy, assessed by percentages of necrotic and residual viable tumor. Secondary endpoint is safety, assessed by adverse events and cosmetic changes. Descriptive statistics will be used. Eligibility criteria Non-pregnant, non-lactating women ≥18 years of age, weighing < 90 kg, who have: histologically proven invasive breast cancer, cT1-2 N0-2 Mx, subtypes invasive ductal carcinoma or invasive carcinoma ‘not otherwise specified’ / ‘no special type’ a tumor in reach of the HIFU beams, diameter ≤ 3.0 cm and distance ≥1.5 cm to skin, nipple and pectoral wall WHO-Performance Score ≤2 a body size fitting in the MR bore and the ability to lie in prone position no prior treatment with neoadjuvant chemotherapy in the past 3 months no prior radiotherapy, thermal therapy or surgery in the targeted breast no contraindications to PSA, MRI or MRI contrast agents no conditions that may interfere with MR-HIFU such as a pacemaker, scar tissue, breast prosthesis or surgical clips no extensive intraductal components, determined on biopsy Additionally, patients may be excluded when the risk of adjuvant over- or undertreatment (due to performing Bloom and Richardson grading on the tumor biopsy) is considered too high. Target accrual 10 patients (present: 0, IRB approval obtained) Conclusion In this phase II trial we aim to show the feasibility of complete tumor ablation of breast cancer with a dedicated MR-HIFU breast system. This approach could lead to a non-invasive treatment alternative for selected patients with primary breast cancer. References 1Merckel 2016, Eur Radiol; 2Deckers 2015, Phys Med Biol; 3Merckel 2013, Cardiovasc Intervent Radiol; Acknowledgements Financial support of Center for Translational Molecular Medicine and Vrienden UMC Utrecht. Profound Medical provides non-financial support. Contact information Josanne de Maar, j.s.demaar@umcutrecht.nl ClinicalTrials.gov Identifier: NCT02407613 Citation Format: Josanne Sophia de Maar, Roel Deckers, Lambertus Wilhelmus Bartels, Thijs van Dalen, Joost van Gorp, Paul J van Diest, Arjen J Witkamp, H. H.B. Vaessen, Maria NicoleGerardineJohanna Aleida Braat, Chrit Moonen. Efficacy of magnetic resonance-guided high intensity focused ultrasound for the ablation of breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT3-07-01.

Start of Antiplatelet Therapy Increases the Prevalence of Intraplaque Hemorrhage in Patients With Advanced Carotid Artery Lesions: A Longitudinal MR Imaging Study

Introduction: Intraplaque hemorrhage (IPH) is an important feature of plaque vulnerability. Patients with a history of transient ischemic attack or stroke are treated with platelet aggregation inhibitors to prevent secondary myocardial infarction or ischemic stroke. Although antiplatelet medication is effective in reduction of clot formation, it also has adverse effects. It has therefore been hypothesized that platelet aggregation inhibitors could also initiate the onset or progression of IPH. A histopathological study on 154 carotid endarterectomy plaques found that preoperative antiplatelet therapy was associated with an increase in the prevalence of repeated IPH in symptomatic carotid plaques. This longitudinal study investigates the effect of antiplatelet agents on the prevalence of IPH. Methods: 63 symptomatic patients underwent carotid MRI at baseline and after two years of follow-up. Presence of IPH in both carotid arteries was scored. Medication prescription before and after the index event and after follow-up was collected.The presence of IPH was determined using MP-RAGE images and analysed blinded for the clinical data using dedicated vessel wall analysis software (VesselMass). IPH was considered present when a hyperintense signal, compared to the sternocleidomastoid muscle, was visible in the bulk of the plaque. Results: A significant higher prevalence of any IPH was found two years after the index event (42.9% vs. 55.6%; p = 0.008) (Figure 1) Patients already using antiplatelet agents before the index event (n=19) did not show significantly more IPH (52.6% vs. 63.2%, p = 0.50) at follow-up. Patients who did not use platelet aggregation inhibitors before the index event (n=44) and were prescribed medication after the event, showed a significantly higher prevalence of IPH (38.6% vs. 52.3%, p=0.031) at follow-up (Figure 2) Conclusion: Patients who started antiplatelet therapy after the index event show a significant increase in bilateral IPH two years after inclusion, while no significant changes in IPH status were found in the patients that already use antiplatelet therapy before the index event. Future studies are warranted to investigate causal relationships. Disclosure: JE Wildberger reports institutional grants from AGFA, Bard, Bayer, GE, Optimed, Philips, Siemens outside the submitted work; and speakers Bureau: Bayer, Siemens outside the submitted work. This research was performed within the framework of the Center for Translational Molecular Medicine (www.ctmm.nl), project PARISk (grant 01C-202), and supported by the Dutch Heart Foundation. This project has received funding from the European Union Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 722609. ME Kooi and JE Wildberger are supported by Stichting de Weijerhorst. ME Kooi is supported by an Aspasia Grant 2018/SGw/00460457 from NWO. J Hendrikse has received funding from the European Research Council under the European Union's Horizon 2020 Programme (H2020) / ERC grant agreement n°637024. FHBM Schreuder is supported by the Dutch Heart Foundation (2012T077).

Novel Stool-Based Protein Biomarkers for Improved Colorectal Cancer Screening: A Case-Control Study.

The fecal immunochemical test (FIT) for detecting hemoglobin is used widely for noninvasive colorectal cancer (CRC) screening, but its sensitivity leaves room for improvement. To identify novel protein biomarkers in stool that outperform or complement hemoglobin in detecting CRC and advanced adenomas. Case-control study. Colonoscopy-controlled referral population from several centers. 315 stool samples from one series of 12 patients with CRC and 10 persons without colorectal neoplasia (control samples) and a second series of 81 patients with CRC, 40 with advanced adenomas, and 43 with nonadvanced adenomas, as well as 129 persons without colorectal neoplasia (control samples); 72 FIT samples from a third independent series of 14 patients with CRC, 16 with advanced adenomas, and 18 with nonadvanced adenomas, as well as 24 persons without colorectal neoplasia (control samples). Stool samples were analyzed by mass spectrometry. Classification and regression tree (CART) analysis and logistic regression analyses were performed to identify protein combinations that differentiated CRC or advanced adenoma from control samples. Antibody-based assays for 4 selected proteins were done on FIT samples. In total, 834 human proteins were identified, 29 of which were statistically significantly enriched in CRC versus control stool samples in both series. Combinations of 4 proteins reached sensitivities of 80% and 45% for detecting CRC and advanced adenomas, respectively, at 95% specificity, which was higher than that of hemoglobin alone (P < 0.001 and P= 0.003, respectively). Selected proteins could be measured in small sample volumes used in FIT-based screening programs and discriminated between CRC and control samples (P < 0.001). Lack of availability of antibodies prohibited validation of the top protein combinations in FIT samples. Mass spectrometry of stool samples identified novel candidate protein biomarkers for CRC screening. Several protein combinations outperformed hemoglobin in discriminating CRC or advanced adenoma from control samples. Proof of concept that such proteins can be detected with antibody-based assays in small sample volumes indicates the potential of these biomarkers to be applied in population screening. Center for Translational Molecular Medicine, International Translational Cancer Research Dream Team, Stand Up to Cancer (American Association for Cancer Research and the Dutch Cancer Society), Dutch Digestive Foundation, and VU University Medical Center.

Classification of patients with sepsis according to blood genomic endotype: a prospective cohort study

Host responses during sepsis are highly heterogeneous, which hampers the identification of patients at high risk of mortality and their selection for targeted therapies. In this study, we aimed to identify biologically relevant molecular endotypes in patients with sepsis. This was a prospective observational cohort study that included consecutive patients admitted for sepsis to two intensive care units (ICUs) in the Netherlands between Jan 1, 2011, and July 20, 2012 (discovery and first validation cohorts) and patients admitted with sepsis due to community-acquired pneumonia to 29 ICUs in the UK (second validation cohort). We generated genome-wide blood gene expression profiles from admission samples and analysed them by unsupervised consensus clustering and machine learning. The primary objective of this study was to establish endotypes for patients with sepsis, and assess the association of these endotypes with clinical traits and survival outcomes. We also established candidate biomarkers for the endotypes to allow identification of patient endotypes in clinical practice. The discovery cohort had 306 patients, the first validation cohort had 216, and the second validation cohort had 265 patients. Four molecular endotypes for sepsis, designated Mars1-4, were identified in the discovery cohort, and were associated with 28-day mortality (log-rank p=0·022). In the discovery cohort, the worst outcome was found for patients classified as having a Mars1 endotype, and at 28 days, 35 (39%) of 90 people with a Mars1 endotype had died (hazard ratio [HR] vs all other endotypes 1·86 [95% CI 1·21-2·86]; p=0·0045), compared with 23 (22%) of 105 people with a Mars2 endotype (HR 0·64 [0·40-1·04]; p=0·061), 16 (23%) of 71 people with a Mars3 endotype (HR 0·71 [0·41-1·22]; p=0·19), and 13 (33%) of 40 patients with a Mars4 endotype (HR 1·13 [0·63-2·04]; p=0·69). Analysis of the net reclassification improvement using a combined clinical and endotype model significantly improved risk prediction to 0·33 (0·09-0·58; p=0·008). A 140-gene expression signature reliably stratified patients with sepsis to the four endotypes in both the first and second validation cohorts. Only Mars1 was consistently significantly associated with 28-day mortality across the cohorts. To facilitate possible clinical use, a biomarker was derived for each endotype; BPGM and TAP2 reliably identified patients with a Mars1 endotype. This study provides a method for the molecular classification of patients with sepsis to four different endotypes upon ICU admission. Detection of sepsis endotypes might assist in providing personalised patient management and in selection for trials. Center for Translational Molecular Medicine, Netherlands.

Abstract 3166: Querying, viewing and analyzing colorectal cancer translational research studies in tranSMART

Abstract All translational research projects basically share the same design, which is correlating variation in disease phenotype to variation in underlying biology. Typical questions to be addressed are: ‘Which (out of thousands) biomarkers predict good/bad outcome?’ and ‘Which (out of thousands) biomarkers predict whether a patient will benefit from a particular therapy?’. In line with this concept, many research teams have generated large amounts of experimental molecular data from patient samples, yet generally this information is inaccessible for examination due to local storage of both (meta)data and the data processing workflows used. Alternatively, data stored in central databases may only be available for exploration and interpretation by data specialists, provided that the processing workflow has been published and is available. Thus, if data is not recorded and easily retrievable, validation of obtained results (e.g. promising biomarkers) may be virtually impossible. Additionally, it will be difficult to query existing data sets to answer new questions, which may lead to experiments being unnecessarily repeated and biological materials being wasted. In the Netherlands, the Translational Research IT (TraIT) project initiated by the Center for Translational Molecular Medicine (CTMM, www.ctmm-trait.nl) aims to provide IT solutions to support translational research from start to end, including sustainable management and analysis of data. These data types involve the clinical, biomedical imaging, biobanking, (molecular) experimental data domains and their associated workflows. In addition to domain-specific solutions to manage these data types, the processed or ‘final’ data of these different domains will become available in the data-integration platform tranSMART for querying, visualization and analysis. To ensure sustainable data stewardship and provide easy access to existing data for the CTMM colorectal cancer project ‘Decrease in Colorectal Cancer Death (DeCoDe)’, data has been made available in tranSMART and more datasets are being added. The data encompasses both clinical and (molecular) experimental data from non-high-throughput molecular profiling (NHTMP) assays and array-based profiling techniques. It is now possible with tranSMART to explore the respective DeCoDe datasets and perform various analyses (survival, Fisher-exact tests, ANOVA, aCGH tests etc.), without needing deep bioinformatics expertise. Through metadata tags it is possible to trace back raw or pre-processed data in other tools (e.g. clinical data in OpenClinica, array-data in GEO, or histological images of tissue microarrays). Data can be examined in more detail by domain experts using tools they are familiar with or other tools provided by CTMM-TraIT. Thus, existing rich data are made findable, accessible, interoperable and reusable (FAIR) and source data can be traced back for customized processing. Citation Format: Mariska Bierkens, Wim van der Linden, Ward Weistra, Kees van Bochove, Jeroen A.M. Beliën, Remond J.A. Fijneman, Rita Azevedo, Jan-Willem Boiten, Gerrit A. Meijer. Querying, viewing and analyzing colorectal cancer translational research studies in tranSMART. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3166.

Multi-Dimensional Impact of the Public-Private Center for Translational Molecular Medicine (CTMM) in the Netherlands: Understanding New 21(st) Century Institutional Designs to Support Innovation-in-Society.

Knowledge translation is at the epicenter of 21st century life sciences and integrative biology. Several innovative institutional designs have been formulated to cultivate knowledge translation. One of these organizational innovations has been the Center for Translational Molecular Medicine (CTMM), a multi-million public-private partnership in the Netherlands. The CTMM aims to accelerate molecular diagnostics and imaging technologies to forecast disease susceptibilities in healthy populations and early diagnosis and personalized treatment of patients. This research evaluated CTMM's impact on scientific, translational, clinical, and economic dimensions. A pragmatic, operationally-defined process indicators approach was used. Data were gathered from CTMM administrations, through a CTMM-wide survey (n = 167) and group interviews. We found that the CTMM focused on disease areas with high human, clinical, and economic burden to society (i.e., oncology, cardiovascular, neurologic, infection, and immunity diseases). CTMM displayed a robust scientific impact that rests 15%-80% above international reference values regarding publication volume and impact. Technology translation to the clinic was accelerated, with >50% of projects progressing from pre-clinical development to clinical testing within 5 years. Furthermore, CTMM has generated nearly 1500 Full Time Equivalent (FTE) of translational R&D capacity. Its positive impact on translational, (future) clinical, and economic aspects is recognized across all surveyed stakeholders. As organizational innovation is increasingly considered critical to forge linkages between life sciences discoveries and innovation-in-society, lessons learned from this study may inform other institutions with similar objectives such as the Clinical and Translational Science Awards (CTSA) Program of the National Institutes of Health (NIH) in the United States.

Sex-specific placental differences as a contributor to sex-specific metabolic programming?

Over the past decades, maternal nutritional intake has been proven to affect the prenatal and post-natal development of the foetus as well as its long-term health (see (Jimenez-Chillaron et al. 2012) and (Lillycrop & Burdge 2015) for recent overviews). In this journal, we have recently shown that maternal Western-style diet gives rise to long lasting programming of the liver in adult mouse offspring (Pruis et al. 2014), affecting mostly the males. Moreover, we demonstrated that several aspects of sex-specific programming are already present in 2-week-old offspring (Mischke et al. 2013), that is, well before puberty. The question arising now is whether this sexual dimorphism was already present during prenatal development, and which factors contributed to its manifestation. One major determinant of development and intrauterine growth is the placental supply of nutrients, which occurs primarily by a combination of diffusion and transporter-mediated transport (Sandovici et al. 2012). The surface area for exchange is a major determinant of these pathways. Despite the important role of the placenta, very few studies have investigated the relation of sex and placental development as a putative explanation of sex-specific programming. Therefore, we here present an overview of the foetal and placental findings in our previously described mouse model (Pruis et al. 2014). To determine the impact of maternal diet and sex on foetal development, C57BL/6J dams were fed either a low-fat diet (10 kcal% from fat, 18 mg cholesterol kg−1) or Western-style high-fat diet (45 kcal% from fat, 196.5 mg cholesterol kg−1) 2 weeks pre-mating and during pregnancy. At gestational day 18.5, dams were killed under isoflurane anaesthesia and foetuses and placentas were characterized. Interestingly, male foetuses exposed to the Western diet during development weighed significantly more than those exposed to low-fat diet, while there was no difference in body length (P = 0.085, not significant) (Fig. 1a,b). In addition, also the placentas of male offspring exposed to Western diet were of a slightly, but significantly larger weight than low-fat diet-fed male offspring (Fig. 1c). In female offspring of these dams, none of these differences due to maternal diet were observed (Fig. 1a–c). Due to the set-up of the experiments, we had only 5 male and 5 female placenta samples for histological examination. However, we observed a distinct difference in placental histology in terms of the ratio between the labyrinth layer and the spongiotrophoblast layer in male offspring (Fig. 1d,e). As mentioned previously, the major determinant of intrauterine growth is the placental supply of nutrients to the foetus. The labyrinthine zone is the main site for nutrient transfer in the mouse placenta during late gestation, and its absolute volume and volume fraction are known to increase progressively during the second half of pregnancy. The estimated depth of the labyrinth layer for the male mice was 872 ± 161 μm, while the measurement for the females was 340 ± 48 μm. As placental layering was affected, mRNA levels of selected transporter genes were measured on gestational day 18.5 by TaqMan real-time PCR. We determined the expression of genes responsible for glucose transport (Glut1 (Slc2a1), Glut4 (Slc2a2), Glut12 (Slc2a12), cholesterol uptake (Ldlr, Scarb1), and cholesterol excretion (Abca1, Abcg1). Neither maternal diet nor sex significantly affected the examined transcription levels (Fig. 1f). In summary, we observed an increase in weight of the male foetuses and their placentas under Western diet conditions. Moreover, there was a distinct difference between the male and female placentas: The larger labyrinth diameter in male placentas compared with the female placentas might lead to a higher transport capacity of nutrients, which let us speculate that the male placenta supports transport of excess maternal nutrients to the male foetus. On the contrary, the female placenta might act as a barrier which limits nutrient supply to the female foetus. As a result, the male foetus shows increased growth under these conditions of maternal surplus nutrients, whereas the female foetus is protected. Our findings are supported by other mouse studies which report that placental changes to prenatal stimuli differ between both sexes, supporting the idea that placentas of female foetuses protect against prenatal insults (Cuffe et al. 2012, 2014, O'Connell et al. 2013). Our findings might contribute to a better understanding of the sex differences observed in many animal studies in the field of long-term metabolic programming. The authors have no conflicts of interest to declare. This research was performed within the framework of CTMM, the Center for Translational Molecular Medicine (www.ctmm.nl), project PREDICCt (grant 01C-104), and supported by the Dutch Heart Foundation, Dutch Diabetes Research Foundation and Dutch Kidney Foundation. TP is supported by the Netherlands Organization for Health Research and Development (ZonMW TOP grant 91211053).

Circulating MicroRNAs Characterize Insufficient Coronary Collateral Artery Development in Patients with Chronic Total Occlusion

Coronary collateral artery growth, arteriogenesis, significantly impacts mortality after an acute coronary event. We aimed to identify microRNAs (miRNAs) that differentiate between insufficient or sufficient coronary collateral artery development in patients with chronic total occlusion (CTO).CTO patients (n=41) were dichotomized based on their pressure‐derived collateral flow index (CFI = (wedge pressure – central venous pressure)/(aortic pressure – central venous pressure)) into low (CFI&lt; 0.39) and high arteriogenic responders (CFI &gt; 0.39). MiRNA profiling on aortic plasma and verification by qPCR, revealed higher levels of miR423‐5p (p &lt; 0.05), miR10b (p &lt; 0.05), miR30d (p &lt; 0.05) and miR126 (p &lt; 0.001) were present in low arteriogenic responders. Receiver operator characteristic curves showed that only miR126 can distinguish patients with low arteriogenic response (area under curve 0.812, p &lt; 0.01, cut‐off 0.037).In conclusion, we have identified miRNAs linked with poor collateralization and found miR126 as the most suitable circulating biomarker to distinguish patients with poor or adequate coronary artery collateral capacity.Supported by the Center for Translational Molecular Medicine (EMINENCE).

Single Sample Application of the EMC92/SKY92 Signature Using the Mmprofiler

IntroductionMultiple Myeloma (MM) is a heterogeneous disease with diverse gene expression patterns (GEP) across patients. This has led to the development of various signatures allowing virtual karyotyping, defining different clusters of patients, and prognostication by high risk signatures (e.g. EMC92/SKY92). Several GEP datasets exist, but may have scaling/offset differences (batch effects) in the data, e.g. due to differences in reagents used, location, etc. Batch wise normalization approaches can reduce batch effects, and have allowed successful validation of those signatures across independent datasets. Batch wise normalization requires groups of patients that have a similar distribution of clinical characteristics, and hence cannot be applied on single patients. Here we demonstrate the validity of applying GEP algorithms on single patients using the MMprofiler, enabling the application of GEP in a routine clinical setting.Materials and MethodsThe MMprofiler GEP assay is a standardized assay from bone marrow to data analysis and result reporting. It was used for 77 MM patients that were enrolled in the HOVON87/NMSG18 trial (73 patients) or HOVON95/EMN02 trial (4 patients). A representative reference set of 30 HOVON patients was selected from which normalization parameters were derived, to be used for normalization of a single sample against this HOVON reference dataset. The remaining 47 samples served as an independent set of samples. In addition, we have also used the publicly available GEP data from 247 patients (MRC-IX trial) as independent samples. This MRC-IX dataset has been produced using different reagents and sample work-up procedures. Therefore, it is likely that a batch effect will exist relative to the HOVON reference dataset, which may influence correctness of single sample analyses.The GEP data from the 47 and 247 independent samples were normalized using two approaches. Firstly, by batch wise mean variance normalization (i.e. across the 47 and 247 patient batches separately). And secondly, by single sample normalization using the normalization parameters from the initial 30 HOVON samples. Subsequently, several classifiers (EMC92/SKY92 etc.) were applied to the data, and their results were compared between the two normalization approaches.ResultsFigure 1 shows the EMC92/SKY92 scores that were obtained after batch normalization (x-axis) and single sample normalization (y-axis). For the 47 HOVON samples there is a high degree of concordance with data points close to the identity line (y=x). Only 2 out of the 47 samples would switch assignment, which is not unexpected since those 2 samples are really close to the threshold (e.g. might also switch due to technical variation). For the MRC-IX dataset, based on single sample normalization more patients would be predicted as high risk (87 (35.2%) instead of 52 (21.0%), see Figure 1), which is caused by a positive offset (i.e. intersect with the y-axis) due to the batch effect.For the Virtual t(4;14) classifier, both datasets have a very high concordance with 0 out 47 HOVON samples, and 5 out of 247 MRC-IX samples (but really close to the threshold) switching assignment (see Figure 1). Hence, even in the presence of a potential batch effect in the MRC-IX dataset, the single sample predictions are accurate. These data suggest that single sample normalization of microarray GEP is possible but requires the strict standardization of the MMprofiler assay and algorithms.ConclusionsScores for the EMC92/SKY92 signature were nearly equivalent when derived from the data following single sample normalization and batch normalization in the Skyline generated data. In the external dataset, a much higher discrepancy was found, highlighting the need to use highly standardized methods to generate Affymetrix GeneChip results. Further validation of this method is planned, and will include replicate runs systematically controlled for various conditions.AcknowledgmentsThis research was performed within the framework of CTMM, the Center for Translational Molecular Medicine, project BioCHIP grant 03O-102. [Display omitted] DisclosuresVan Vliet:SkylineDX: Employment. Dumee:SkylineDx: Employment. de Best:SkylineDx: Employment. Sonneveld:SkylineDx: Membership on an entity’s Board of Directors or advisory committees. van Beers:SkylineDX: Employment.

Prognostic and Predictive Gene Expression Profiling (GEP) Markers Confirmed in Carfilzomib, Lenalidomide, and Dexamethasone (KRd) Treated Newly Diagnosed Multiple Myeloma (NDMM) Patients (Pts)

Introduction High rates of complete response (CR) have previously been demonstrated in KRd-treated NDMM pts in a phase 1/2 trial (trial 1; NCT01029054) and a phase 2 trial that combined KRd with autologous stem cell transplant (ASCT) (trial 2; NCT01816971). Here, we report results of extended follow-up from the 2 trials and correlate response and PFS with GEP performed using the MMprofiler GEP assay, which provides results for the SKY92 signature, 7 virtual karyotyping markers (t[4;14], t[11;14], t[14;16]/t[14;20], gain[1q], del[13q], del[17p], H-MM [virtual gain(9q)]), and 3 clusters (MF, MS, CD2). Previously, a combination of 5 markers (SKY92, virtual gain[1q], virtual t[14;16]/t[14;20], MF and CD2 clusters) was identified that predicts improved outcomes when treated with the proteasome inhibitor (PI) bortezomib (van Vliet et al, EHA 2014). We evaluated the SKY92 prognostic signature, virtual karyotyping markers, and this 5-marker PI predictor signature in order to confirm these markers as predictive signatures in the KRd setting. Materials and Methods In the consecutive trials 1 and 2, pts received 4 cycles of KRd induction, followed by extended KRd treatment with deferred ASCT (trial 1; NCT01029054) or ASCT followed by extended KRd treatment (trial 2; NCT01816971). In both trials, pts received single-agent lenalidomide as maintenance after completion of KRd. The MMprofiler GEP assay was performed on RNA from CD138+ purified plasma cells. As depth of response with KRd is associated with improved time-to-event outcomes (Jasielec et al, ASH 2013), data were analyzed for associations between any of the MMprofiler markers and the groups that achieved ≥near CR (nCR) vs Results Based on a July 1, 2014 cutoff date, 91 pts were enrolled (53 in trial 1; 38 in trial 2). CD138+ RNA was available from 27 pts (16 from trial 1; 11 from pts in trial 2 who completed ≥4 KRd cycles). Pts with H-MM (virtual gain[9q]) were significantly less likely to achieve nCR (p=0.027) (Table 1). None of the other markers were found to have a statistically significant association with response; several markers, however, showed a trend for predictive value at the current sample size. PFS data were available for 16 pts (median follow-up: trial 1, 40 months [mo]; trial 2, 6.7 mo [with no events at the cutoff date]). Two markers were found to be significantly prognostic (Figure 1): SKY92 (5/16 pts [31%]; HR=5.7; p=0.023) and virtual gain (1q) (6/16 pts [38%]; HR=5.5; p=0.017). Virtual t(4;14) was marginally significant (HR=3.7, p=0.055). When considering the 5-marker PI predictor signature, none of the 16 pts were positive for virtual t(14;16)/t(14;20), MF or CD2 clusters. Combining the SKY92 and virtual gain(1q) markers identified a highly significant subgroup of high-risk pts (7/16; 44%) with a median PFS of 26 mo (HR=8.7; p=0.0088; Figure 1). In no-risk pts (9/16), estimated 4-year PFS was 78%. Conclusions Absence of H-MM (virtual gain[9q]) identified pts more likely to achieve ≥nCR by the end of 4 cycles of KRd, with other markers awaiting validation from ongoing enrollment to the KRd + ASCT trial. Based on combined SKY92 high-risk and virtual gain(1q) signatures, the MMprofiler assay detected a highly significant group of high-risk pts with inferior PFS compared to the no-risk group. Despite the inferior outcome, these high-risk pts appeared to have benefited from KRd when compared to historical data of treatment without PIs, suggesting that the 5-marker PI predictor signature (Van Vliet et al, EHA 2014) may also apply to carfilzomib-based treatment. We observed a 4-year PFS rate in no-risk pts in the KRd trials which trended higher than the historical outcome of the PAD arm in the HOVON65/GMMG-HD4 trial (78% vs 34%, respectively), suggesting a benefit for KRd without ASCT in this group of pts compared with other strategies with PIs; further study is needed, however. With ongoing enrollment into the KRd + ASCT trial, the findings reported here will be further validated. Updated results will be presented at the meeting. Acknowledgments This research was performed within the framework of CTMM, the Center for Translational Molecular Medicine, project BioCHIP grant 03O-102. Disclosures van Vliet:SkylineDx: Employment. Vij:Celgene: Honoraria, Research Funding; Onyx: Honoraria, Research Funding; Sanofi: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Millennium: Honoraria; Array: Honoraria. Dumee:SkylineDx: Employment. Bosman:SkylineDx: Employment. deBest:SkylineDx: Employment. van Beers:SkylineDx: Employment. Jakubowiak:Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Millennium: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; SkylineDX: Honoraria, Membership on an entity9s Board of Directors or advisory committees.

Abstract 4935: Hypoxia targeting fluorescent nanobodies for optical molecular imaging of preinvasive breast cancer

Abstract Introduction: Optical molecular imaging is a novel strategy in diagnosis and therapy of breast cancer. Essential for succesful implementation of this technology is a high tumor-to-normal tissue ratio (TNR), which requires tumor-specific probes/tracers. However, most tumor markers are expressed in a minority of tumors only. Therefore, we have developed a novel probe binding to the membrane-bound protein carbonic anhydrase IX (CAIX), which is commonly expressed in solid tumors as a result of hypoxic conditions. The probe is based on nanobodies (15 kDa, variable domains obtained from heavy-chain antibodies present in animals from the Camelidae family), that has a faster tumor uptake and body clearance than conventional (monoclonal) antibodies, and can be applied for molecular imaging of pre-invasive breast cancer. Methods: High affinity CAIX-specific nanobodies were selected using a modified phage display technology and site-directed conjugated to IRDye800CW. Mice bearing orthotopically transplanted MCF10DCIS (DCIS) and CAIX-overexpressing MCF10DCIS (DCIS+CAIX) xenografts were injected with fluorescent CAIX-specific nanobody (B9-IR), or non-specific control nanobody (R2-IR) and imaged up to 48 hours non-invasively. Tumors were resected under image-guidance. Nanobody uptake was assessed in tissue sections and quantified using a biodistribution assay. Results: A highly specific nanobody for CAIX was obtained. In DCIS+CAIX xenografts, mean TNR was 4.3±0.6 (8 mice) already two hours after injection. In DCIS tumors, mean TNR was 1.8±0.1 (8 mice), which was higher than TNR obtained after injection with R2-IR (1.4±0.2, 4 mice). After three hours, probe accumulation was sufficient to allow imag-guided tumor resection. Biodistribution studies showed that B9-IR uptake was 4.6±0.8% of injected dose per gram in DCIS tumor tissue (%I.D./g), while 2.0±0.2 %I.D./g was found with R2-IR. Upon fluorescence imaging of tissue sections, B9-IR was present in perinecrotic areas, while R2-IR showed no accumulation in these regions. Conclusion: We have developed a novel IRDye800CW-labeled anti-CAIX-nanobody that can be used for rapid optical molecular imaging of (pre-)invasive breast cancer before and during surgery. Furthermore, the stability of the conjugate allowed for ‘molecular fluorescence pathology’, which might results in better contrast than conventional chromagen based immunohistochemistry at the pathology department. Acknowledgement: This research was supported by the Center for Translational Molecular Medicine (MAMMOTH). Citation Format: Aram SA van Brussel, Arthur Adams, Sabrina Oliveira, Mohamed El Khatabbi, Jeroen F. Vermeulen, Elsken Van der Wall, Willem PThM Mali, Patrick WB Derksen, Paul J. Van Diest, Paul MP Van Bergen en Henegouwen. Hypoxia targeting fluorescent nanobodies for optical molecular imaging of preinvasive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4935. doi:10.1158/1538-7445.AM2014-4935

Abstract 3005: Vascular endothelial growth factor A - a systematic review and meta-analysis of expression patterns in breast cancer

Abstract Background Vascular Endothelial Growth Factor-A (VEGF-A), pivotal for neo-angiogenesis, is an interesting target for breast cancer molecular imaging. To assess its potential clinical value, we performed a systematic review and meta-analysis of VEGF-A expression rates in normal breast tissue, benign and (pre-)invasive breast disease, and investigated associations with clinicopathological characteristics. Methods We systematically searched EMBASE and MEDLINE for articles describing VEGF-A expression in breast disease assessed by immunohistochemistry (IHC) or Enzyme-Linked Immunosorbent Assay (ELISA). We pooled IHC expression rates with random-effects models and applied meta-regression to identify associations with clinicopathological characteristics. We summarized findings for ELISA results. Results Of 2,711 unique articles retrieved, 157 studies described IHC results (16,046 tissue samples). Pooled expression rates were lower in studies applying antibody clone VG1 (-21%, p=0.018) or C-1 (-24%, p=0.016), compared to A-20. Expression compared to invasive carcinoma was lower in normal tissue (-51%, p&amp;lt;0.001) and benign breast diseases (-34%, p&amp;lt;0.001), but comparable for carcinoma in situ (+5%, p=0.289). Expression rates increased with tumour grade (II: +14%, p&amp;lt;0.001; III: +25%, p&amp;lt;0.001), stage (II: +11%, p&amp;lt;0.001; III: +22%, p&amp;lt;0.001), size (T3/4: +11%, p=0.004), HER2-positivity (+14%, p&amp;lt;0.001), lymph node positivity(+11%, p&amp;lt;0.001), and decreased in hormone receptor positive (PR-positive: -7%, p&amp;lt;0.001; ER-positive:-8%, p&amp;lt;0.001) and lobular cancers (-14%, p&amp;lt;0.001). Substantial heterogeneity was observed in study results. Furthermore, expression rates strongly depended on VEGF-A positivity threshold. VEGF-A levels assessed by ELISA (42 studies, 8,498 samples) were highly variable. Conclusion Although reported VEGF-A expression rates in breast cancer were variable, we found significant correlations with clinicopathological characteristics indicating aggressive disease. Also, expression rates were low in normal breast tissue and benign breast disease. As expression rates strongly depended on VEGF-A positivity - arguing against an on/off phenomenon - applicability of VEGF-A for molecular imaging purposes will depend on sensitivity of the molecular imaging modality, target population, and envisioned applications. Acknowledgment This research was supported by the Center for Translational Molecular Medicine (MAMMOTH). Citation Format: Arthur Adams, Jeroen Vermeulen, Peter Zuithoff, Elsken Van der Wall, Laetitia Lamberts, Elisabeth de Vries, Johannes de Jong, Gooitzen Van Dam, Paul J. van Diest, Willem P.Th.M. Mali, Sjoerd G. Elias. Vascular endothelial growth factor A - a systematic review and meta-analysis of expression patterns in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3005. doi:10.1158/1538-7445.AM2014-3005

SAT0088 Economic Evaluation of A Tight-Control Treatment Strategy Using an Imaging Device (HANDSCAN) for Monitoring Joint Inflammation in Early Rheumatoid Arthritis

BackgroundNowadays treat-to-target by means of tight-control is generally accepted as the standard treatment principle in early rheumatoid arthritis (RA) (Schoels et.al.). However, this requires frequent (monthly) visits including comprehensive clinical...

Temporal response of monocytes during progressive coronary artery occlusion (1071.7)

Elevated levels of circulating monocytes (MCs) have been correlated with collateral development in coronary artery disease patients. The aim of this study was to employ a model of progressive coronary artery occlusion (CO), in order to compare the temporal response of MC upregulation in the systemic circulation vs. their infiltration into ischemic myocardium.An ameroid constrictor was implanted on the left coronary artery of New Zealand White rabbits (CO after 7‐10 days). Peripheral blood MCs were isolated, fluorescently labelled and administered intravenously, 9 (n=4),16 (n=4) or 28 (n=5) days after surgery. Coronary vessels were perfused with fluorescent cast. Hearts were processed with an imaging cryomicrotome, generating 3D reconstructions of the vascular network and MCs.Peripheral blood MCs increased 83.47±55.18% 9 days after CO surgery, while 16 and 28 days after CO surgery showed 39.96±65.27 and ‐13.82±24.17 % change, respectively. MC subset ratios (classical/non‐classical) showed the largest percentage increase after 16 days CO in peripheral blood. In the myocardium, MC infiltration was localized in the ischemic area, and was highest after 16 days vs. 9 days CO (0.027±0.023 vs. 0.017±0.001 of total heart volume, p&lt;0.05). Collateral connections coincided with MC localization. In conclusion, we note that the temporal MC response in the systemic circulation may differ from that in the myocardium.Grant Funding Source: Supported by the Center for Translational Molecular Medicine (EMINENCE).

A1.7 Serum cytokine changes in rituximab-treated rheumatoid arthritis patients

BackgroundAlthough B cell depletion therapy with rituximab is successful in the majority of rheumatoid arthritis (RA) patients, 30–40% of patients do not respond. The mechanism behind this is yet unknown,...

Increased FXIa Levels In Patients With a First Acute Myocardial Infarction: The Introduction Of a New Thrombin Generation Based FXIa Assay

IntroductionAcute myocardial infarction (AMI) is still an important cause of morbidity and mortality. One of the contributing mechanisms in AMI is plasma hypercoagulability, with recent studies suggesting activation of factor (F) XI to play a role in the onset of the thrombotic event. By serving as an amplification loop that enhances thrombin generation (TG), FXIa is thought to be involved in thrombotic diseases. To quantify FXIa levels in plasma, we developed a new TG based assay and hypothesized that in AMI patients FXIa plasma levels are increased at the time of the thrombotic event, as compared to 6 months after and compared to healthy controls. MethodsA prospective cohort study was performed including patients with a first AMI. Citrated blood samples were collected upon admission (before administration of heparin) and after 6 months. Citrated blood samples from apparently healthy individuals served as a control population. FXIa TG was measured by means of the Calibrated Automated Thrombogram. All plasma samples were diluted at a 1:5 ratio in FXI deficient plasma and triggering was by addition of 4 µM phospholipids (without tissue factor). Active site-inhibited factor VII (30 nM) was added to inhibit the extrinsic coagulation pathway. FXIa plasma concentrations were established using a FXIa reference curve, which was obtained by addition of varying concentrations of FXIa (0-100 pM) to a corn trypsin inhibitor (CTI) pool-FXI deficient plasma mixture. The established FXIa concentrations were related to the 1-year outcome: cardiovascular death, recurrent MI, a second coronary intervention and ischemic stroke. ResultsA total of 56 consecutive AMI patients (71% men), with a mean age on admission of 60 (SD ±12) years, were included. The healthy control population consisted of 30 subjects (67% men) with a mean age of 50 (SD ±6) years. FXIa plasma concentrations were significantly increased in AMI patients at day 0 compared to 6 months after the event (4.8 pM [3.6-6.9] vs. 3.9 pM [2.4-5.7], median ± IQR; P=0.0015), and also compared to the FXIa levels in apparently healthy subjects (4.8 pM [3.6-6.9] vs. 3.8 pM [2.4-4.9], median ± IQR; P=0.0075). No significant difference in FXIa concentrations was demonstrated between AMI patients at 6 months follow-up and apparently healthy controls. In the 12-month follow-up period, 11 patients reached an endpoint. However, FXIa concentrations in the highest quartile were not significantly associated with the development of a recurrent thrombotic cardiovascular event (OR 3.2 [95%CI 0.4-28.2], P=0.29). ConclusionsThis study presents the first application of a new TG based FXIa assay. Results showed that in AMI patients at the time of the thrombotic event there is a significantly increased FXIa plasma concentration, compared to 6 months after the event and compared to healthy controls. However, no significant association could be established between the FXIa concentration and the risk of recurrent ischemic cardiovascular complications. AcknowledgmentThis research was supported by the Center for Translational Molecular Medicine and the Dutch Heart Foundation (INCOAG). Disclosures:No relevant conflicts of interest to declare.

High Risk Multiple Myeloma Cases Are Identified In An MMRC Led Study By The SKY92 Gene Signature (MMprofiler)

IntroductionMultiple Myeloma is not a single disease. There is increasing support for risk classification in combination with treatment decision making because of its impact on clinical outcomes. Here we demonstrate additional evidence of the prognostic value of SKY92, an established genetic marker of high risk Multiple Myeloma in a multicenter collection of samples with undisclosed treatments. Materials MethodsA public GEP dataset (MMRC, MMGI portal) contained 114 cases of untreated Multiple Myeloma and was used for SKY92 high risk OS prediction (Kuiper et al. Leukemia 2012). In collaboration with MMRC, OS (with a minimum of at least 2 year follow-up) was collected for 91 of 114 cases for the purpose of this analysis. Briefly, CD138-positive plasma cells had been purified prior to total RNA extraction and subsequent gene expression profiling on Affymetrix U133Plus2.0 GeneChips. The 91 cases represented 9 different clinical sites and their CEL files were normalized as a single batch against a reference cohort of 329 cases after which the SKY92 risk scores were determined as either standard risk or high risk. ResultsSKY92 resulted in 19 high risk (20.9%) versus 72 standard risk (79.1%) cases in the unselected 91 case-cohort. Comparisons with other high risk GEP signatures will be performed. The OS analysis (Figure 1) shows that the HR cases have significantly shorter survival (Hazard Ratio 11, p = 7 x 10-5). Table 1 shows that high risk patients had more elevated B2M (26.3% vs 13.9%), more low albumin (26.3% vs 16.7%) and more high creatinine (26.3% vs 11.0%). There was no difference between high and standard groups in diagnosis dates (not shown). Cause of the 16 (84.2%) deaths among the high risk cases, and 21 (29.1%) deaths among the standard risk cases indicates that high risk contains less disease progression deaths (57.1% vs 31.3%), and more unknown deaths (56.3% vs 23.8%). [Display omitted] [Display omitted] ConclusionsThe SKY92 classifier identified 19 of 91 cases (21%) as high risk, recapitulating the percentage of high risk in previously studied cohorts (Kuiper et al. 2012). Moreover the hazard ratio of 11 when events up to 24 months or 8.18 when all events are considered, emphasizes the unmet medical need of high risk cases identified with SKY92 as 69% of all deaths within 2 years (9/13 death events) were in this category. AcknowledgmentsThis research was performed within the framework of CTMM, the Center for Translational Molecular Medicine, project BioCHIP grant 03O-102. Rafael Fonseca is a Clinical Investigator of the Damon Runyon Cancer Research Fund. This work is supported by grants R01 CA83724, ECOG CA 21115T, Predolin Foundation, Mayo Clinic Cancer Center and the Mayo Foundation. Disclosures:van Beers:Skyline Diagnostics: Employment. Van Vliet:Skyline Diagnostics: Employment. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership. Jagannath:Celgene: Honoraria; Millennium: Honoraria. Jakubowiak:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kumar:Celgene: Clinical Trial Support Other, Membership on an entity's Board of Directors or advisory committees; Cephalon: Clinical Trial Support, Clinical Trial Support Other; Millennium: Clinical Trial Support, Clinical Trial Support Other, Membership on an entity's Board of Directors or advisory committees; Novartis: Clinical Trial Support, Clinical Trial Support Other; Onyx: Clinical Trial Support Other, Membership on an entity's Board of Directors or advisory committees. Lebovic:Celgene: Speakers Bureau; Onyx: Speakers Bureau. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy. Reece:Onyx: Honoraria; Novartis: Honoraria; Millennium: Research Funding; Merck: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Research Funding. Siegel:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Vij:Celgene: Honoraria, Research Funding, Speakers Bureau; Millennium: Honoraria, Speakers Bureau; Onyx: Honoraria, Research Funding, Speakers Bureau. Zimmerman:Celgene: Honoraria; Millennium: Honoraria; Onyx: Honoraria. Fonseca:Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy, Research Funding; Binding Site: Consultancy; Millennium: Consultancy; AMGEN: Consultancy; Cylene: Research Funding; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties.

Comparison Of Conventional, FISH and GEP Prognostic Factors In Multiple Myeloma: Introducing a Novel Risk Stratification

IntroductionMultiple Myeloma (MM) is a heterogeneous disease with highly variable survival. Gene expression profiling (GEP) classifiers, such as the EMC-92, can consistently distinguish high risk patients from standard risk patients. Other prognostic factors for MM include the international staging system (ISS) and FISH. Here we present a comparison of prognostic factors and introduce a novel stratification based on EMC-92 and ISS. MethodsScores were calculated for the GEP classifiers EMC-92, UAMS-70, UAMS-17, UAMS-80 and MRC-IX-6 for the following five studies: HOVON-65/GMMG-HD4 (n=328; GSE19784), MRC-IX (n=247; GSE15695), UAMS-TT2 (n=345; GSE2658), UAMS-TT3 (n=238; E-TABM-1138 and GSE2658) and APEX (n=264; GSE9782; for details, see Kuiper R, et al. Leukemia (2012) 26: 2406–2413). FISH data were available for the HOVON-65/GMMG-HD4 trial and the MRC-IX trial. ISS values were available for all datasets except UAMS-TT2. Univariate associations between markers and overall survival (OS) were investigated in a Cox regression analysis, using Bonferroni multiple testing correction. For pair wise analysis of markers, the significance in the increase of partial likelihood was calculated. In order to find the strongest combination (defined as the highest partial likelihood) of GEP-ISS, we compared these pair-wise on the same data. Training sets of classifiers were excluded for those analyses in which that specific classifier was tested. All survival models have been stratified for study. The calculations were done in R using the package survival. ResultsPrognostic value of FISH, GEP and serum markers was determined in relation to overall survival (Figure 1). GEP classifiers generally performed much better than FISH markers. Of 6 FISH markers with known adverse risk, del(17p), t(4;14), t(14;20) and del(13q) demonstrated a significant association only in one of two data sets with available FISH (HOVON-65/GMMG-HD4). GEP classifiers, on the other hand, are much more robust. Classifiers EMC-92, UAMS-70 and UAMS-80 significantly identify a high-risk population in all evaluated data sets, whereas the UAMS-17 and the MRC-IX-6 classifiers predict high-risk patients in three of four datasets. As expected, ISS staging demonstrated stable and significant hazard ratios in most studies (three out of four). Indeed, when evaluating a merged data set, both ISS and all evaluated GEP classifiers are strong prognostic factors independent of each other. Markers with additive value to each other include all combinations of GEP classifiers as well as the combination of GEP classifiers together with ISS. Tested in independent sets, the EMC-92 classifier combined with ISS is the best combination, as compared to other classifier-ISS combinations tested on the same independent data sets. The strongest risk stratification in 3 groups was achieved by splitting the EMC-92 standard risk patients into low risk, based on ISS stage I, and intermediate risk, based on ISS stage II and III. This stratification retains the original EMC-92 high-risk group, and is robust in all cohorts. The proportions of patients defined as low, intermediate and high risk for this combined EMC-92-ISS classifier are 31% / 47% / 22 % (HOVON-65/GMMG-HD4), 19% / 61% / 20 % (MRC-IX), 46% / 39% / 15 % (UAMS-TT3) and 32% / 55% / 13 % (APEX). Variability in low risk proportion is caused by the variable incidence of ISS stage I. [Display omitted] ConclusionsWe conclude that GEP is the strongest predictor for survival in multiple myeloma and far more robust than FISH. Adding ISS to EMC-92 results in the strongest combination of any of the GEP classifier-ISS combinations. Stratification in low risk, intermediate and high risk may result in improved treatment and ultimately in longer survival of MM patients.This research was supported by the Center for Translational Molecular Medicine (BioCHIP project) Disclosures:van Vliet:Skyline Diagnostics: Employment. Mulligan:Millennium Pharmaceuticals: Employment. Morgan:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Millenium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Johnson and Johnson: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Goldschmidt:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria. van Beers:Skyline Diagnostics: Employment. Sonneveld:Janssen-Cilag: Honoraria; Celgene: Honoraria; Onyx: Honoraria; Janssen-Cilag: Research Funding; Millenium: Research Funding; Onyx: Research Funding; Celgene: Research Funding.

OP0020 Validation of gene signatures to predict rheumatoid arthritis development

BackgroundEarly recognition of development of rheumatoid arthritis (RA) allows timely start of treatment. It is known that antibodies against citrullinated proteins (ACPA) and rheumatoid factor (RF) have a predictive value...