University Hospital Bonn Research Articles

The Junior Research Academy 2017 of the German Research Foundation (DFG) - Promotion of Young Researchers in Health Services

The fourth Junior Research Academy in Health Services Research was once again funded by the German Research Foundation (DFG) in 2017. The academy was initiated by the Centre for Health and Society (chs) in Düsseldorf with the participation of the Centre for Health Services Research Cologne (ZVFK), the Research Centre for Health Communication and Health Services Research (CHSR) of the Clinic and Polyclinic for Psychosomatic Medicine and Psychotherapy of the University Hospital Bonn, the Interdisciplinary Centre for Health Services Research (IZVF) in Witten and the Institute for Health Services Research and Clinical Epidemiology (IVE) of the University Marburg. The aims of the Junior Research Academy are for imparting skills in the development and elaboration of innovative project ideas, to increase the number of proposals from the field of health services research to the DFG, to strengthen basic research within health services research in Germany and to network the scientific community. Young researchers from all over Germany were eligible to apply for participation by submitting an application in the form of a research proposal. A total of 83 applications were received. The 21 most promising applicants (14 women and 7 men) were selected in a 2-stage review process; 20 of these completed the Junior Research Academy program. After a one-day preparatory workshop, the preparation and review of a sample application, an academy week and a finalisation phase, all applications were submitted to the DFG on time. Of these proposals, 9 were funded. The first alumni meeting of the DFG Junior Research Academy took place in Düsseldorf in July 2019.

Comprehensive analysis of tumor necrosis factor receptor TNFRSF9 (4-1BB) DNA methylation with regard to molecular and clinicopathological features, immune infiltrates, and response prediction to immunotherapy in melanoma.

BackgroundImmunotherapy, including checkpoint inhibition, has remarkably improved prognosis in advanced melanoma. Despite this success, acquired resistance is still a major challenge. The T cell costimulatory receptor TNFRSF9 (also known as 4-1BB and CD137) is a promising new target for immunotherapy and two agonistic antibodies are currently tested in clinical trials. However, little is known about epigenetic regulation of the encoding gene. In this study we investigate a possible correlation of TNFRSF9 DNA methylation with gene expression, clinicopathological parameters, molecular and immune correlates, and response to anti-PD-1 immunotherapy to assess the validity of TNFRSF9 methylation to serve as a biomarker.MethodsWe performed a correlation analyses of methylation at twelve CpG sites within TNFRSF9 with regard to transcriptional activity, immune cell infiltration, mutation status, and survival in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas. Furthermore, we used quantitative methylation-specific PCR to confirm correlations in a cohort of N = 115 melanoma patients’ samples (UHB validation cohort). Finally, we tested the ability of TNFRSF9 methylation and expression to predict progression-free survival (PFS) and response to anti-PD-1 immunotherapy in a cohort comprised of N = 121 patients (mRNA transcription), (mRNA ICB cohort) and a case-control study including N = 48 patients (DNA methylation, UHB ICB cohort).FindingsWe found a significant inverse correlation between TNFRSF9 DNA methylation and mRNA expression levels at six of twelve analyzed CpG sites (P ≤ 0.005), predominately located in the promoter flank region. Consistent with its role as costimulatory receptor in immune cells, TNFRSF9 mRNA expression and hypomethylation positively correlated with immune cell infiltrates and an interferon-γ signature. Furthermore, elevated TNFRSF9 mRNA expression and TNFRSF9 hypomethylation correlated with superior overall survival. In patients receiving anti-PD-1 immunotherapy (mRNA ICB cohort), we found that TNFRSF9 hypermethylation and reduced mRNA expression correlated with poor PFS and response.InterpretationOur study suggests that TNFRSF9 mRNA expression is regulated via DNA methylation. The observed correlations between TNFRSF9 DNA methylation or mRNA expression with known features of response to immune checkpoint blockage suggest TNFRSF9 methylation could serve as a biomarker in the context of immunotherapies. Concordantly, we identified a correlation between TNFRSF9 DNA methylation and mRNA expression with disease progression in patients under immunotherapy. Our study provides rationale for further investigating TNFRSF9 DNA methylation as a predictive biomarker for response to immunotherapy.FundingAF was partly funded by the Mildred Scheel Foundation. SF received funding from the University Hospital Bonn BONFOR program (O-105.0069). DN was funded in part by DFG Cluster of Excellence ImmunoSensation (EXC 1023). The funders had no role in study design, data collection and analysis, interpretation, decision to publish, or preparation of the manuscript; or any aspect pertinent to the study.

Questionnaire based evaluation of the motivation of surgically treated patients to participate in preventative hygiene measures.

Background: Nosocomial infections caused by antibiotic-resistant pathogens demonstrate the continued need for preventive hygiene management strategies. Information and training of patients in their personal hygiene is a current requirement of the German Society for Hospital Hygiene, and is recommended by the Commission for Hospital Hygiene and Infection Prevention (Kommission für Krankenhaushygiene und Infektionsprävention beim Robert-Koch Institut, KRINKO) at the Robert Koch Institute.Aim: The aim of this study was to evaluate patients’ existing knowledge of hygiene and their motivation to actively participate in preventive hygiene measures.Methods: This study included 445 inpatients at the Polyclinic for Surgery of University Hospital Bonn. Subjects were interviewed over a 6-month period using a questionnaire comprising 21 questions on the topic of hygiene.Results: The majority of patients rated their subjective level of knowledge as intermediate (41%), 25% as poor and 35% as high. The respondents rated the active inclusion of patients in hygiene practices as highly relevant, and were willing to actively contribute to infection prevention, whereby the patients considered hand washing and hand disinfection in particular as important starting points. 78% of the respondents wanted more information on hygiene, particularly on wound and food hygiene. Targeted hygiene education provided by hospital staff had a positive effect on the patients’ subjective level of information, as well as on their confidence in physicians and nursing staff. Previous information via television or radio had a negative impact on the patients’ subjective information level and on their confidence in hospital staff. Conclusion: Most surgically treated patients are motivated to actively contribute to preventive hygiene measures. This represents an additional and important option for applying hospital hygiene more effectively and, above all, closer to the patient. Information and education should preferably be performed by healthcare professionals.

Epilepsy Identification Based on VMD, RELIEFF Algorithm and Machine Learning Classification Techniques

Epilepsy identification is done by visual observation of electroencephalography (EEG) signals, which is more sensitive to bias and time consuming. In most of the previous research of epileptic seizure detection suffers from unsuitability and low power for processing large datasets. To eliminate aforementioned problems a computerized detection method is required to aid medical professionals. In this paper, a new technique is proposed to identify the epilepsy based on VMD, RELIEFF algorithm and machine learning approach. To investigate the proposed method performance a public EEG dataset is adopted from university hospital bonn, Germany. The technique starts with the VMD, which is used to extract the features from each EEG signal. And then RELIEFF algorithm is adopted to identify the best features. Finally to categorize the normal and epilepsy EEG signals a machine learning classification (ANN, KNN, and SVM) approach is used. The results demonstrate that the adopted method (VMD+RELIEFF+SVM) can achieve a better accuracy, shows that a commanding method to identification and classification of epileptic seizures.

Reconstruction of the skull base in spontaneous rhinoliquorrhea.

Objective/Hypothesis: Spontaneous rhinoliquorrhea often occurs due to defects of the skull base. It is often misinterpreted as rhinitis and is surgically the most difficult rhinoliquorrhea entity to close.Methods: We conducted a retrospective chart analysis of patients that were diagnosed with spontaneous rhinoliquorrhea at the University Hospital Bonn between 2001 and 2017.Results: Overall, twelve patients were included in this study. On average, the time between occurrence of nasal discharge and diagnoses of rhinoliquorrhea was 123 days. In ten patients, the localization of the skull base defect could be localized by computed tomography or MRI cisternography. Ten patients underwent surgery, of which 9 remained recurrence free. One patient underwent revision surgery and from thereon was recurrence free.Conclusion: Spontaneous rhinoliquorrhea still remains a diagnostic and therapeutic challenge. Whenever persistent watery nasal discharge appears in a patient, rhinoliquorrhea must be considered. Endoscopic surgical reconstruction of the skull base is the therapeutic gold standard and should be attempted as soon as the diagnosis is secured.

Systematic expression analysis of the mitochondrial respiratory chain protein subunits identifies COX5B as a prognostic marker in clear cell renal cell carcinoma.

To analyze the expression of mitochondrial respiratory chain protein subunits in clear cell renal cell carcinoma. Possible prognostic candidates were determined using The Cancer Genome Atlas database (n=605). The database provided valid messenger ribonucleic acid expression data for 93 genes encoding for the subunits. Selected subunits were further investigated at the messenger ribonucleic acid and protein level by real-time polymerase chain reaction, western blot and immunohistochemistry with the cohorts of the University Hospital Bonn. The Cancer Genome Atlas messenger ribonucleic acid expression data indicated univariate and multivariate prognostic impact for seven subunits (NDUFS8, NDUFS7, COX5B, COX6B1, SDHD, COX15 and COX19). Using real-time polymerase chain reaction, significant downregulation (P<0.05, n=74) could be shown for COX5B, COX6B1, NDUFS7 and NDUF8 in clear cell renal cell carcinoma tissue. Survival analysis of polymerase chain reaction data showed a non-significant relationship (P=0.067) of high COX5B expression and poor overall survival. Western blot (n=8) and immunohistochemistry analysis (n=167) confirmed significant COX5B downregulation on the protein level. Immunohistochemistry analysis identified COX5B as a prognostic marker for overall (P=0.017) and cancer-specific survival (P=0.042). The present study findings suggest downregulation of additional subunits of mitochondrial respiratory chain proteins in clear cell renal cell carcinoma. Remarkably, COX5B, a subunit of the respiratory chain complexIV, can be identified as a novel prognostic marker.

Patients' priorities in a reminiscence and legacy intervention in palliative care.

Background:Reminiscence is used in a range of different interventions in palliative care, for example, Dignity Therapy or Life Review. However, literature has focused mainly on the methodology, and little has been published on patients’ priorities and primary concerns.Objective:This study looks at themes emerging in a reminiscence intervention with patients confronted with a life-limiting disease. Interviews were audiotaped and transcribed verbatim. Transcripts were analysed using thematic analysis.Setting/subjects:Seventeen patients who were receiving palliative care at the University Hospital Bonn participated in interviews reviewing parts or phases of their lives.Results:Patients expressed satisfaction and a sense of well-being with the intervention. Major themes emerging in the interviews were the factors involved in the development and expression of personality, such as character-forming influences, self-image, self-awareness, and philosophy of life. Talking about personality was entangled with influences from growing up, qualification/job, partner/spouse, children, resources, twists of fate/crossroads, and coping.Conclusion:The topics emerging from the interviews differed from the scope of guiding questions in common reminiscence methods like Life Review or Dignity Therapy. The underlying motivation of patients seemed to be the search for identity and continuity in one’s life.

Radionuclide intake risks in the clinical administration of 223RaCl2

An intake monitoring program covering more than half a year of clinical administration of Radium-223-dichloride for the palliative treatment of castration-resistant prostate cancer was carried out in the nuclear medicine department of the university hospital Bonn. Radioactivity in a total of 87 samples of gloves, air filters, faecal bioassays and face masks was measured and evaluated to assess the need for radiation protection measures for the medical staff. The main aim was to quantify or obtain an upper limit for the intake factor. An intake factor of 10−8 was measured when the preparation of patient doses took place in part in a laminar flow cabinet, which indicates an intake factor of 10−7 in more commonplace practice without a cabinet. The intake factor is therefore at the same level as other standard applications of unsealed sources in nuclear medicine. Our findings confirmed that masks are not required under any circumstances. However, the investigation also revealed that contamination risks, especially during the preparation of doses in syringes, should not be neglected.

Editorial introductions

Editorial introductions

Interprofessional two-man team approach for interhospital transport of ARDS-patients under extracorporeal membrane oxygenation: a 10\u2009years retrospective observational cohort study

BackgroundExtra Corporeal Membrane Oxygenation (ECMO) has become an accepted treatment option for severely ill patients. Due to a limited availability of ECMO support therapy, patients must often be transported to a specialised centre before or after cannulation. According to the ELSO guidelines, an ECMO specialist should be present for such interventions. Here we describe the safety and efficacy of a reduced team approach involving one anaesthesiologist, experienced in specialised intensive care medicine, and a specialised critical care nurse.MethodsThis study is a 10 years retrospective, single institution analysis of all data collected between January 2007 and December 2016 from the medical records at the University Hospital Bonn, Germany.ResultsThe Bonner mobile ECMO team was deployed in 170 cases for on-site evaluation for ECMO support therapy. 4 (2.4%) patients died prior to arrival or during the implementation of ECMO support. Of the remaining 166 patients, 126 were cannulated at the referring site, 40 were transported without ECMO. Of those, 21 were subsequently cannulated out our centre. 19 patients never received ECMO treatment. The primary indication for ECMO treatment was ARDS (159/166 patients). Veno-venous ECMO was initiated in 137, whilst 10 patients received veno-arterial ECMO treatment. Mean transportation time was 75 ± 36 min, and mean transport distance was 56 ± 57 km. In total, 26 complications were observed, three being directly transport-related. The overall survival was 55%.ConclusionsInitiation of extracorporeal membrane oxygenation and subsequent transport can be safely and efficiently performed by a two-man team with good outcome.

68Ga-PSMA PET/CT for monitoring response to 177Lu-PSMA-617 radioligand therapy in patients with metastatic castration-resistant prostate cancer.

To evaluate the use of 68Ga-PSMA PET/CT for monitoring response to 177Lu-617 PSMA radioligand therapy in patients with metastatic castrate-resistant prostate cancer (mCRPC). Patients from the University Hospital Bonn and the University Hospital Aachen were retrospectively reviewed for this study. We included 48 patients with mCRPC who were treated with 177Lu-PSMA-617 and whose records included 68Ga-PSMA PET/CT imaging before the first and after the third or fourth treatment cycle. A treatment response based on 68Ga-PSMA PET/CT was defined according to a modified version of the PERCIST criteria. A decline in PSA level of ≥50% was considered the reference standard. The sensitivity, specificity, positive and negative predictive values, and ROC curves were calculated, and patient survival times in relation to the PET results were also analysed. 68Ga-PSMA PET/CT had a sensitivity of about 85% and a specificity of between 55% and 65%. The negative and positive predictive values ranged between 70% and 78%. The fitted ROC area was 0.70. The survival time was about 19.6months in patients with a treatment response, while nonresponders had a survival time of about 15.9months. However, this difference between the groups was not statistically significant. Our results indicate that 68Ga-PSMA PET/CT could be a useful tool for the evaluation of response to 177Lu-PSMA-617 radioligand therapy within a theranostic framework.

Treatment of HIV in Pregnancy - Progress Over One Decade

Worldwide, 37 million people are infected with HIV; more than 50% are women. Currently, MTCT (mother-to-child transmission) can be reduced to<1%. The intention of the present study was to analyze the development of (1) the course of pregnancy of HIV-infected women, (2) the mode of delivery and (3) the post-exposure prophylaxis of the newborn over the last decade. In this retrospective study, data from HIV-infected women who between 2005 and 2016 received care at the HIV outpatient department and gave birth at the Department of Obstetrics at University Hospital Bonn was analyzed. Furthermore, neonatal data was collected and HIV-MTCT was evaluated. In the 2005-2016 study period, 87 pregnancies in 61 women were identified. Seventy babies were born alive at the Department of Obstetrics, University Hospital Bonn. 53% of these women were of African origin. The median of CD4+ cell count was 510 cells/ml (IQR 444); however, 32 women (52%) had more than 500 cells/ml. During the antenatal period, the HI viral load had been suppressed completely in 77% of women (<50 HIV-1-RNA copies/ml) and was<400 HIV-1-RNA copies/ml in 92% of women. The elective cesarean section rate fell significantly from 77% in the years 2005-2011 to 58% in 2012-2016. The proportion of deliveries after 37 weeks of gestation increased markedly from 60% to 69% after 2012. Additionally, while between 2005-2011 the birth weight of 78% of the newborns was between the 10th and 90th percentile, this proportion increased to 92% after 2012. Fifty-four of 70 newborns (77%) were classified as having low to normal HIV transmission risk. A vertical HIV transmission from mother to child did not occur (0/70). Between 2005 and 2016 no vertical HIV transmission from mother to child occurred (0/70). Due to the change in treatment strategy, the elective cesarean section rate fell significantly as well the rate of premature births. An optimal interdisciplinary collaboration builds the basis for successful treatment of HIV in pregnancy.

The Role of Adding Somatostatin Analogues to Peptide Receptor Radionuclide Therapy as a Combination and Maintenance Therapy.

Purpose: Although somatostatin analogues (SSA) and peptide receptor radionuclide therapy (PRRT) are validated therapies in patients with advanced gastroenteropancreatic neuroendocrine tumors (GEP-NET), it remains unclear whether SSA combined with PRRT or as maintenance therapy can provide prolonged survival compared with patients treated with PRRT alone. In this retrospective study, we aimed to investigate whether there is a survival benefit to adding SSA to PRRT as a combination therapy and/or maintenance therapy.Patients and Methods: The investigation included 168 patients with unresectable GEP-NETs treated at the University Hospital Bonn, Bonn, Germany. The patients were divided into two main groups: PRRT monotherapy (N = 81, group 1) and PRRT plus SSA (N = 87, group 2) as combined therapy with PRRT and/or as maintenance therapy after PRRT.Results: Data for overall survival (OS) were available from 168 patients, of whom 160 had data for progression-free survival (PFS). The median PFS was 27 months in group 1 versus 48 months in group 2 (P = 0.012). The median OS rates were 47 months in group 1 and 91 months in group 2 (P < 0.001). The death-event rates were lower in group 2 (26%) than in group 1 (63%). SSA as a combination therapy with PRRT and/or as a maintenance therapy showed a clinical benefit rate (objective response or stable disease) of 95%, which was significantly higher than group 1 (79%).Conclusions: SSA as a combination therapy and/or maintenance therapy may play a significant role in tumor control in patients with GEP-NET who underwent a PRRT. Clin Cancer Res; 24(19); 4672-9. ©2018 AACR.

Cross-sectional increase of adherence to multidisciplinary tumor board decisions

BackgroundCancer research has made great progress in the recent years. With the increasing number of options in diagnosis and therapy the implementation of tumorboards (TUBs) has become standard procedure in the treatment of cancer patients. Adherence tests on tumor board decisions are intended to enable quality assurance and enhancement for work in tumor boards in order to continuously optimize treatment options for cancer patients.MethodsSubject of this study was the adherence of the recommendations made in three of 14 tumorboards, which take place weekly in the Center for Integrated Oncology (CIO) at the University Hospital Bonn. In total, therapy recommendations of 3815 patient cases were checked on their implementation. A classification into four groups has been made according to the degree of implementation. A second classification followed regarding the reasons for differences between the recommendation and the therapy which the patient actually received.ResultsThe study showed that 80.1% of all recommendations in the three TUBs were implemented. 8.3% of all recommendations showed a deviance. Most important reasons for the deviances were patient wish (36.5%), patient death (26%) and doctoral decision, due to the patient’s comorbidities or side effects of the treatment (24.1%).Interestingly, deviance in all three tumor boards in total significantly decreased over time.ConclusionsAim of the study was to clarify the use of tumor boards and find approaches to make them more efficient. Based on the results efficiency might be optimized by increased consideration of patients` preferences, improved presentation of patient-related data, more detailed documentation and further structuring of the tumor board meetings.

Editorial introductions

Editorial introductions

PD-L1 (CD274) and PD-L2 (PDCD1LG2) promoter methylation is associated with HPV infection and transcriptional repression in head and neck squamous cell carcinomas.

BackgroundDNA methylation of the immune checkpoint gene PD-L1 has recently been shown to be associated with PD-L1 mRNA expression in various malignancies. This study aimed to investigate the association of PD-L1 and PD-L2 methylation with mRNA expression, immune cell infitration, protein expression and human papilloma virus (HPV) infection in head and neck squamous cell carcinoma (HNSCC) patients.ResultsDNA methylation of PD-L1 and PD-L2 correlates inversely with mRNA expression (PD-L1: p ≤ 0.002; PD-L2: p ≤ 0.014). Methylation of specific CpG-sites of both PD-L1 and PD-L2 were further significantly associated with HPV infection in the TCGA cohort. Immune cell infiltrates correlated significantly with PD-L1 and PD-L2 methylation. In the validation cohort, PD-L1 protein expression was associated with PD-L1 hypomethylation (p = 0.012).ConclusionsDNA methylation of PD-L1 and PD-L2 is associated with transcriptional silencing and HPV infection in HNSCCs. Additional studies are warranted to test PD-L1 and PD-L2 methylation as predictive biomarkers for response to immunotherapies (e.g. pembrolizumab and nivolumab) that target the PD-L1/PD-L2/PD-1 immune checkpoint axis.Materials and MethodsPD-L1 and PD-L2 promoter methylation and its mRNA expression were analyzed based on Infinium HumanMethylation450 BeadChip and RNA-Seq (both Illumina, Inc.) data in a representative HNSCC patient cohort (n = 528) enrolled by The Cancer Genome Atlas (TCGA) Research Network. A validation cohort consisting of 168 HNSCC patients treated at the University Hospital Bonn was analyzed regarding PD-L1 and PD-L2 promoter methylation by means of methylation-specific quantitative real-time PCR. PD-L1 protein expression in the validation cohort was quantified via immunohistochemistry (PD-L1 antibody clone 22C3, Dako/Agilent Technologies, Inc.).

Automatic Epileptic Seizure Detection in EEG Signals Using Multi-Domain Feature Extraction and Nonlinear Analysis

Epileptic seizure detection is commonly implemented by expert clinicians with visual observation of electroencephalography (EEG) signals, which tends to be time consuming and sensitive to bias. The epileptic detection in most previous research suffers from low power and unsuitability for processing large datasets. Therefore, a computerized epileptic seizure detection method is highly required to eradicate the aforementioned problems, expedite epilepsy research and aid medical professionals. In this work, we propose an automatic epilepsy diagnosis framework based on the combination of multi-domain feature extraction and nonlinear analysis of EEG signals. Firstly, EEG signals are pre-processed by using the wavelet threshold method to remove the artifacts. We then extract representative features in the time domain, frequency domain, time-frequency domain and nonlinear analysis features based on the information theory. These features are further extracted in five frequency sub-bands based on the clinical interest, and the dimension of the original feature space is then reduced by using both a principal component analysis and an analysis of variance. Furthermore, the optimal combination of the extracted features is identified and evaluated via different classifiers for the epileptic seizure detection of EEG signals. Finally, the performance of the proposed method is investigated by using a public EEG database at the University Hospital Bonn, Germany. Experimental results demonstrate that the proposed epileptic seizure detection method can achieve a high average accuracy of 99.25%, indicating a powerful method in the detection and classification of epileptic seizures. The proposed seizure detection scheme is thus hoped to eliminate the burden of expert clinicians when they are processing a large number of data by visual observation and to speed-up the epilepsy diagnosis.

LDB1 overexpression is a negative prognostic factor in colorectal cancer.

BackgroundColorectal cancer (CRC) is the third most common cancer in western countries and is driven by the Wnt signaling pathway. LIM-domain-binding protein 1 (LDB1) interacts with the Wnt signaling pathway and has been connected to malignant diseases. We therefore aimed to evaluate the role of LDB1 in CRC.ResultsOverexpression of LDB1 in CRC is associated with strikingly reduced overall and metastasis free survival in all three independent patient cohorts. The expression of LDB1 positively correlates with genes involved in the Wnt signaling pathway (CTNNB1, AXIN2, MYC and CCND1). Overexpression of LDB1 in CRC cell lines induced Wnt pathway upregulation as well as increased invasivity and proliferation. Upon separate analysis, the role of LDB1 proved to be more prominent in proximal CRC, whereas distal CRC seems to be less influenced by LDB1.Materials and MethodsThe expression of LDB1 was measured via RT-qPCR in 59 clinical tumor and normal mucosa samples and correlated to clinical end-points. The role of LDB1 was examined in two additional large patient cohorts from publicly available microarray and RNAseq datasets. Functional characterization was done by lentiviral overexpression of LDB1 in CRC cell lines and TOP/FOP, proliferation and scratch assays.ConclusionsLDB1 has a strong role in CRC progression, confirmed in three large, independent patient cohorts. The in vitro data confirm an influence of LDB1 on the Wnt signaling pathway and tumor cell proliferation. LDB1 seems to have a more prominent role in proximal CRC, which confirms the different biology of proximal and distal CRC.

PITX3 promoter methylation is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients after radical prostatectomy.

BackgroundMolecular biomarkers that might help to distinguish between more aggressive and clinically insignificant prostate cancers (PCa) are still urgently needed. Aberrant DNA methylation as a common molecular alteration in PCa seems to be a promising source for such biomarkers. In this study, PITX3 DNA methylation (mPITX3) and its potential role as a prognostic biomarker were investigated. Furthermore, mPITX3 was analyzed in combination with the established PCa methylation biomarker PITX2 (mPITX2).MethodsmPITX3 and mPITX2 were assessed by a quantitative real-time PCR and by means of the Infinium HumanMethylation450 BeadChip. BeadChip data were obtained from The Cancer Genome Atlas (TCGA) Research Network. DNA methylation differences between normal adjacent, benign hyperplastic, and carcinomatous prostate tissues were examined in the TCGA dataset as well as in prostatectomy specimens from the University Hospital Bonn. Retrospective analyses of biochemical recurrence (BCR) were conducted in a training cohort (n = 498) from the TCGA and an independent validation cohort (n = 300) from the University Hospital Bonn. All patients received radical prostatectomy.ResultsIn PCa tissue, mPITX3 was increased significantly compared to normal and benign hyperplastic tissue. In univariate Cox proportional hazards analyses, mPITX3 showed a significant prognostic value for BCR (training cohort: hazard ratio (HR) = 1.83 (95 % CI 1.07–3.11), p = 0.027; validation cohort: HR = 2.56 (95 % CI 1.44–4.54), p = 0.001). A combined evaluation with PITX2 methylation further revealed that hypermethylation of a single PITX gene member (either PITX2 or PITX3) identifies an intermediate risk group.ConclusionsPITX3 DNA methylation alone and in combination with PITX2 is a promising biomarker for the risk stratification of PCa patients and adds relevant prognostic information to common clinically implemented parameters. Further studies are required to determine whether the results are transferable to a biopsy-based patient cohort. Trial registration: Patients for this unregistered study were enrolled retrospectively.

Abstract 4388: The role of neoantigens in immunotherapy of cutaneous melanoma

Abstract Melanoma is increasing in incidence by up to 3% annually, and metastatic melanoma, which is resistant to most conventional therapies, has a poor prognosis with a 5-year survival rate of less than 20%. Immunotherapy is able to yield durable response in melanoma; however, the response rate is limited (20-30%). It has been shown that tumor immunogenicity is highly correlated to the response to immunotherapy. The mutational load and the characteristics of the neoantigens displayed by the tumor cells are theorized to play an important role in the immunogenicity of the tumor. We used genetically engineered preclinical mouse models to study the role of these neoantigens in melanoma response to immune therapy. Three syngeneic C57BL/6 mouse melanoma models were developed: UV induced melanoma in albino BRAF(V600E);PTEN-/- mice (1), DMBA induced melanoma in pigmented HGF-tg;CDK4(R24C) mice (2), and UV induced melanoma in pigmented HGF-tg mice. These three models were found to have varying degrees of tumor immunogenicity based on vaccination studies. The UV induced BRAF(V600E);PTEN-/- model displayed poor immunogenicity, while the DMBA induced HGF-tg;CDK4(R24C) model exhibited moderate immunogenicity and the UV induced HGF-tg model showed the highest immunogenicity. Exome sequencing results showed that high immunogenicity is associated with mutations of genes in DNA damage responses and frameshift mutations. To test the role of neoantigens, RNA from each of the melanoma models was sequenced and analyzed for mutational and expression profiles. We will be comparing the response of these three mouse models to correlate immunogenicity with response to anti-CTLA-4 therapy. We plan to analyze the pathways in which mutated genes were enriched, allowing the identification of “druggable” targets to enhance therapeutic efficacy of immune checkpoint inhibitors. CRISPR/Cas9-based screening approaches will be used to identify specific neoantigens able to influence response to immune therapy. In conclusion, our preclinical mouse models show that tumor immunogenicity may be correlated to the carcinogenic mechanisms, and RNA sequence has provided further insight into the role of mutational load and neo-epitopes on immune-based therapeutic response. (1) Provided by Marcus Bosenberg (Yale School of Medicine, New Heaven, CT), Martin McMahon (Huntsman Cancer Institute, Salt Lake City, UT). (2) Provided by Thomas Tueting (University Hospital Bonn, Bonn, Germany). Citation Format: Renee M. Thomas, Chi-Ping Day, Zoe Weaver Ohler, Rajaa El Meskini, Carri Graff-Cherry, Sung Chin, Aleksandra Michalowski, Ji Luo, Terry Van Dyke, Glenn Merlino. The role of neoantigens in immunotherapy of cutaneous melanoma. [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 4388.