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Secondary, or functional, mitral regurgitation (FMR) was recently recognized as a separate clinical entity, complicating heart failure with reduced ejection fraction (HFrEF) and entailing particularly poor outcome. Currently, there is a lack of targeted therapies for FMR due to the fact that pathomechanisms leading to FMR progression are incompletely understood. In this study, we sought to perform metabolomic profiling of HFrEF patients with severe FMR, comparing results to patients with no or mild FMR.Targeted plasma metabolomics and untargeted eicosanoid analyses were performed in samples drawn from HFrEF patients (n = 80) on optimal guideline-directed medical therapy. Specifically, 17 eicosanoids and 188 metabolites were analysed. Forty-seven patients (58.8%) had severe FMR, and 33 patients (41.2%) had no or non-severe FMR. Comparison of eicosanoid levels between groups, accounting for age, body mass index, and sex, revealed significant up-regulation of six eicosanoids (11,12-EET, 13(R)-HODE, 12(S)-HETE, 8,9-DiHETrE, metPGJ2, and 20-HDoHE) in severe FMR patients. Metabolites did not differ significantly. In patients with severe FMR, but not in those without severe FMR, levels of 8,9-DiHETrE above a cut-off specified by receiver-operating characteristic analysis independently predicted all-cause mortality after a median follow-up of 43 [interquartile range 38, 48] months [hazard ratio 12.488 (95% confidence interval 3.835-40.666), P 0.0001].We report the up-regulation of various eicosanoids in patients with severe FMR, with 8,9-DiHETrE appearing to predict mortality. Our observations may serve as a nucleus for further investigations into the causes and consequences of metabolic derangements in this important valvular abnormality.

Purpose: Although prognostic and predictive factors in ovarian cancer have been extensively studied for decades, only few have been identified and introduced to clinical practice. Here, we evaluate hVps37A (HCRP1) as a possible novel predictive marker for ovarian cancer. hVps37A was originally described as a member of the membrane-trafficking ESCRT-I complex mediating the internalization and degradation of ubiquitinated membrane receptors.Experimental Design: We analyzed an ovarian cancer tissue microarray for HCRP1, EGFR, and HER2 expression. We used a tetracycline inducible ovarian cancer cell culture model to show the effects of hVps37A knockdown in vitro and in vivo. In addition, we studied the effects of epidermal growth factor receptor (EGFR) inhibitors cetuximab and lapatinib on ovarian cancer cells under conditions of hVps37A knockdown.Results: We find that hVps37A is significantly downregulated in ovarian cancer and modifies the prognostic value of EGFR and HER2 expression. In addition, hVps37A downregulation in ovarian cancer cells leads to cytoplasmic pEGFR retention and hyperactivation of downstream pathways and is associated with enhanced xenograft growth in nude mice and invasion of the collagen matrix. Furthermore, due to subsequent sustained Akt- and MAPK-pathway activation, hVps37A-deficient cells become irresponsive to inhibition by the therapeutic antibody cetuximab.Conclusion: We propose that hVps37A status could become a novel prognostic and therapeutic marker for EGFR or HER2 driven tumors. Clin Cancer Res; 17(24); 7816–27. ©2011 AACR.

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Log2 concentrations of all 89 GPhLs. Subdivided into healthy controls (blue) and each the four most extreme patients (green, with the highest and red, with the lowest GPhL concentrations), sorted from top to bottom according concentration levels. Significant OS-associated GPhLs are labeled in bold.

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Purpose: Cancer metabolism is characterized by alterations including aerobic glycolysis, oxidative phosphorylation, and need of fuels and building blocks.Experimental Design: Targeted metabolomics of preoperative and follow-up sera, ascites, and tumor tissues, RNA sequencing of isolated tumor cells, local and systemic chemokine, and local immune cell infiltration data from up to 65 high-grade serous ovarian cancer patients and 62 healthy controls were correlated to overall survival and integrated in a Systems Medicine manner.Results: Forty-three mainly (poly)unsaturated glycerophospholipids and four essential amino acids (citrulline) were significantly reduced in patients with short compared with long survival and healthy controls. The glycerophospholipid fingerprint is identical to the fingerprint from isolated (very) low-density lipoproteins (vLDL), indicating that the source of glycerophospholipids consumed by tumors is (v)LDL. A glycerophospholipid-score (HR, 0.46; P = 0.007) and a 100-gene signature (HR, 0.65; P = 0.004) confirmed the independent impact on survival in training (n = 65) and validation (n = 165) cohorts. High concentrations of LDLs and glycerophospholipids were independently predictors for favorable survival. Patients with low glycerophospholipids presented with more systemic inflammation (C-reactive protein and fibrinogen negatively and albumin positively correlated) but less adaptive immune cell tumor infiltration (lower tumor and immune cell PD-L1 expression), less oxygenic respiration and increased triglyceride biosynthesis in tumor cells, and lower histone expressions, correlating with higher numbers of expressed genes and more transcriptional noise, a putative neo-pluripotent tumor cell phenotype.Conclusions: Low serum phospholipids and essential amino acids are correlated with worse outcome in ovarian cancer, accompanied by a specific tumor cell phenotype. Clin Cancer Res; 23(8); 2081–92. ©2016 AACR.

Figure S1. Scheme of used methods (top box) and samples (colored bars) and obtained results; Figure S2. Correlations of median serum concentrations of metabolite classes of 62 healthy controls and 65 HGSOC patients; Figure S3. Boxplot of median concentrations of metabolite classes and glutamine and sum of hexoses (mainly glucose) divided in healthy controls and groups of different three year survivals; Figure S5. Boxplots of overall survival association-values of 19 amino acids, grouped according different physicochemical characteristics or according essential and non-essential for humans (red box); Figure S6. Median concentrations of GPhLs with different degrees of unsaturation in controls and patients with different three year survivals; Figure S7. Correlations of the GPhL score with nutritional measures, body mass index (BMI), and the Eastern Cooperative Oncology Group (ECOG) performance status; Figure S8. Correlations of the GPhL score ("Ascites"), low density protein (LDL), and the mean concentrations of other metabolite groups (non-significant glycerophospholipids (GPhLs), significant and non-significant with OS associated amino acids (AAs), and acylcarnitines) with ascites. (p-values, trend tests); Figure S9. Correlations of the GPhL score with the percentages of PD-L1 expressing tumor cells, PDL1 expressing tumor infiltrating immune cells, and intraepithelial CD8 positive immune cells; Figure S10. Histograms of correlation coefficients between serum and ascites GPhL concentrations; Figure S11. Targeted metabolomics of tumor tissues and correlations with matched serum samples; Figure S12. Targeted metabolomics of follow-up sera from 19 patients; Figure S13. KEGG pathways in HGSOC cancer cells, significantly associated with the GPhL score; Figure S14. Cyto- and chemokines correlated to the GPhL score in serum and ascites; Table S1. Characteristics of all high grade serous ovarian cancer patients and sub-cohorts used for various analyses and of healthy controls; Table S2. Univariate (corrected for tissue type) and multiple linear regression analyses between the GPhL score and immune checkpoint measures; Table S3. SPIA pathway analysis with 2,054 significant genes (FDR

Supplementary Figure Legends 1-4, Table 1 from Oncogenic BARD1 Isoforms Expressed in Gynecological Cancers

Supplementary Figures 1-4 from Oncogenic BARD1 Isoforms Expressed in Gynecological Cancers

BARD1 is required for protein stability and tumor suppressor functions of BRCA1, which depend on the ubiquitin ligase activity of the BRCA1-BARD1 heterodimer. The NH2-terminal RING domains of both proteins act as interaction modules and form a ubiquitin ligase, which has functions in DNA repair, cell cycle checkpoint regulation, and mitosis. Interestingly, up-regulated expression of truncated BARD1 isoforms was found to be associated with poor prognosis in breast and ovarian cancers and, in a hormonally regulated fashion, in the human cytotrophoblast, a cell type with properties reminiscent of cancer cells. We therefore performed reverse transcription-PCR to determine the structure of BARD1 isoforms in cell lines derived from hormone-dependent and hormone-independent cancers. We found a specific combination of isoforms, generated by differential splicing and alternative transcription initiation, mostly lacking the BRCA1 interaction domain, in gynecologic but not hematologic cancer cell lines. To investigate the prevalence of BARD1 isoforms in tumors, we applied immunohistochemistry to ovarian cancers, using antibodies distinguishing full-length BARD1 and isoforms. Expression of NH2 terminally truncated BARD1 was correlated with advanced stage of cancer, and expression of spliced isoforms was typical for clear cell carcinoma, the ovarian cancer with worst prognosis, suggesting a role of BARD1 isoforms in cancer progression. To challenge this hypothesis, we silenced BARD1 isoforms in ovarian cancer cells that lacked wild-type BARD1 by siRNA interference, which led to a complete proliferation arrest. Thus, BARD1 isoform expression is required for cancer cell proliferation, which is compatible with the notion that BARD1 isoforms act as cancer maintenance genes. [Cancer Res 2007;67(24):11876–85]

Introduction Ulcerative colitis (UC) is a chronic disease with rising incidence and unclear etiology. Deep molecular phenotyping by multi omics analyses may provide novel insights into disease processes and characteristic features of remission states. Methods UC pathomechanisms were assessed by proteome profiling of human tissue specimen, obtained from five distinct colon locations for each of the 12 patients included in the study. Systemic disease-associated alterations were evaluated thanks to a cross-sectional setting of mass spectrometry-based multi-omics analyses comprising proteins, metabolites and eicosanoids of plasma obtained from UC patients during acute episodes and upon remission in comparison to healthy controls. Results Tissue proteome profiling indicated colitis-associated activation of neutrophils, macrophages, B- and T-cells, fibroblasts, endothelial cells and platelets, and hypoxic stress, and suggested a general down-regulation of mitochondrial proteins accompanying the establishment of apparent wound healing-promoting activities including scar formation. While pro-inflammatory proteins were apparently upregulated by immune cells, the colitis-associated epithelial cells, fibroblasts, endothelial cells and platelets seemed to predominantly contribute anti-inflammatory and wound healing-promoting proteins. Blood plasma proteomics indicated chronic inflammation and platelet activation, whereas plasma metabolomics identified disease-associated deregulations of gut and gut microbiome-derived metabolites. Upon remission, several, but not all, molecular candidate biomarker levels recovered back to normal. Conclusions The findings may indicate that microvascular damage and platelet deregulation hardly resolve upon remission but apparently persist as disease-associated molecular signature. This study presents local and systemic molecular alterations integrated in a model for UC pathomechanisms potentially supporting the assessment of disease and remission states in UC patients.

Ovarian cancer (OC) is the most lethal gynecological malignancy with a 5-year survival rate of 49%. This is caused by late diagnosis when cells have already metastasized into the peritoneal cavity and to the omentum. OC progression is dependent on the availability of high-energy lipids/fatty acids (FA) provided by endogenous de novo biosynthesis and/or through import from the microenvironment. The blockade of these processes may thus represent powerful strategies against OC. While this has already been shown for inhibition of FA/lipid biosynthesis, evidence of the role of FA/lipid import/transport is still sparse. Therefore, we treated A2780 and SKOV3 OC cells with inhibitors of the lipid uptake proteins fatty acid translocase/cluster of differentiation 36 (FAT/CD36) and low-density lipoprotein (LDL) receptor (LDLR), as well as intracellular lipid transporters of the fatty acid-binding protein (FABP) family, fatty acid transport protein-2 (FATP2/SLC27A2), and ADP-ribosylation factor 6 (ARF6), which are overexpressed in OC. Proliferation was determined by formazan dye labeling/photometry and cell counting. Cell cycle analysis was performed by propidium iodide (PI) staining, and apoptosis was examined by annexin V/PI and active caspase 3 labeling and flow cytometry. RNA-seq data revealed altered stress and metabolism pathways. Overall, the small molecule inhibitors of lipid handling proteins BMS309403, HTS01037, NAV2729, SB-FI-26, and sulfosuccinimidyl oleate (SSO) caused a drug-specific, dose-/time-dependent inhibition of FA/LDL uptake, associated with reduced proliferation, cell cycle arrest, and apoptosis. Our findings indicate that OC cells are very sensitive to lipid deficiency. This dependency should be exploited for development of novel strategies against OC.

PARP inhibitors (PARPi) have increased treatment options in ovarian cancer, particularly in patients with BRCA1/2 mutations, although there are still marked differences in the duration of patients’ response to this targeted therapy. BRCA testing is routinely performed in tumor tissue of ovarian cancer patients. The resulting molecular pathological findings include the genetic nomenclature of the mutation, the frequency of the mutated allele (variant allele frequency, VAF), and the tumor cell content. VAF measures the percentage of mutated alleles from the total alleles in the cells of the examined tissue. The aim of this study was to investigate the significance of VAF on the therapeutic response to PARPis in ovarian cancer patients. Epithelial ovarian cancer patients harboring BRCA1/2 tumor mutations, who underwent germline testing and received PARPi therapy at the Medical University of Vienna (n = 41) were included in the study. Corrected VAF (cVAF) was calculated based on VAF, tumor cell content, and germline mutation. Patients were divided into two groups based on their cVAF. Median PFS under PARPi in patients with low cVAF was 13.0 months (IQR [10.3-not reached]) and was not reached in the high cVAF group. High cVAF was significantly associated with longer PFS in the multivariate analysis (HR = 0.07; 95% CI [0.01–0.63]; p = 0.017). In conclusion, high cVAF was associated with a significantly better response to PARPi in this study population.

The therapeutic potential of immune checkpoint inhibitors is currently being investigated in epithelial ovarian cancer (EOC), but immunological effects of the programmed cell death protein 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis in EOC still remain poorly understood. The aim of this study was thus to compare infiltration rates of PD-1 and PD-L1 expressing tumor infiltrating leucocytes (TILs) in primary ovarian tumor tissue and metastatic intraperitoneal implants and to investigate its impact on overall survival (OS). Tumor specimens (ovarian tumor tissues and intraperitoneal metastases) of 111 patients were used to investigate the PD-1, PD-L1 and CD8 expression rates on TILs and PD-L1 expression rate of tumor cells. The percentages of CD8, PD-1, and PD-L1 expressing subpopulations of TILs differ in primary ovarian tumor tissues and metastatic intraperitoneal implants. High PD-1 among TILs in peritoneal metastases were associated with favorable OS. High PD-L1 expression in TILs was associated with poor OS. Combining both factors in peritoneal metastases revealed an unfavorable prognosis. Primary ovarian tumor tissue and intraperitoneal metastatic tissues in EOC might have different strategies to evade immune control. Those findings are of importance for the process of biomarker assessment to predict patients’ response to immunotherapy.

Background Accumulating evidence links brown adipose tissue (BAT) to increased cold-induced energy expenditure (CIEE) and regulation of lipid metabolism in humans. BAT has also been proposed as a novel source for biologically active lipid mediators including polyunsaturated fatty acids (PUFAs) and oxylipins. However, little is known about cold-mediated differences in energy expenditure and various lipid species between individuals with detectable BAT positive (BATpos) and those without BAT negative (BATneg). Methods Here we investigated a unique cohort of matched BATpos and BATneg individuals identified by 18F-fluorodeoxyglucose positron emission tomography combined with computed tomography ([18F]-FDG PET/CT). BAT function, CIEE, and circulating oxylipins, were analyzed before and after short-term cold exposure using [18F]-FDG PET/CT, indirect calorimetry, and high-resolution mass spectrometry, respectively. Results We found that active BAT is the major determinant of CIEE since only BATpos individuals experienced significantly increased energy expenditure in response to cold. A single bout of moderate cold exposure resulted in the dissipation of an additional 20 kcal excess energy in BATpos but not in BATneg individuals. The presence of BAT was associated with a unique systemic PUFA and oxylipin profile characterized by increased levels of anti-inflammatory omega-3 fatty acids as well as cytochrome P450 products but decreased concentrations of some proinflammatory hydroxyeicosatetraenoic acids when compared with BATneg individuals. Notably, cold exposure raised circulating levels of various lipids, including the recently identified BAT-derived circulating factors (BATokines) DiHOME and 12-HEPE, only in BATpos individuals. Conclusions In summary, our data emphasize that BAT in humans is a major contributor toward cold-mediated energy dissipation and a critical organ in the regulation of the systemic lipid pool.

Discrimination between benign and malignant adnexal masses is essential for optimal treatment planning, but still remains challenging in a routine clinical setting. In this retrospective study, we aimed to compare albumin as a single parameter to calculate models by analyzing laboratory parameters of 1552 patients with an adnexal mass (epithelial ovarian cancer (EOC): n= 294; borderline tumor of the ovary (BTO): n = 66; benign adnexal mass: n = 1192) undergoing surgery. Models comprising classical laboratory parameters show better accuracies (AUCs 0.92–0.93; 95% CI 0.90–0.95) compared to the use of single markers, and could easily be implemented in clinical practice by containing only readily available markers. This has been incorporated into a nomogram.

Prostate-specific membrane antigen (PSMA) is present in the tumor-associated neovasculature of many cancer types. Current data in ovarian cancer are limited and controversial; thus, the aim of this study was to investigate PSMA expression in a larger and homogenous patient cohort. This might lead to further studies investigating the use of imaging and therapeutic modalities targeting PSMA. Eighty patients with advanced stage high-grade serous ovarian cancers were included. Using immunohistochemistry, PSMA and CD31, a marker for endothelial cells, were examined in whole tissue sections. Percentage and intensity of PSMA expression were determined in the neovasculature. Expression levels were correlated with clinicopathological parameters and survival. Low (≤10%), medium (20–80%), and high (≥90%) PSMA expression was found in 14, 46, and 20 ovarian cancer samples, respectively. PSMA expression was confined to tumor-associated neovasculature and significantly correlated with progression-free (HR 2.24, 95% CI 1.32–3.82, p = 0.003) and overall survival (HR 2.73, 95% CI 1.41–5.29, p = 0.003) in multivariate models, considering age, FIGO stage, and residual disease. This is the first study showing a clinical relevance for PSMA in patients with ovarian cancer. PSMA was detected in the vast majority of cancer samples and showed an impact on survival.

e15604 Background: Immune checkpoint inhibitors (ICI) do not seem to be effective in solid cancers, which lack an immunogenic priming. Radiotherapy (RT) can induce an immunogenic cell death (ICD) and thereby restore the susceptibility to ICI, especially in microsatellite stable (MSS) cancers. This study evaluates safety, tolerability and feasibility of neoadjuvant chemoradiotherapy (CRT) with concomitant ipilimumab (IPI) and nivolumab (NIVO) in locally advanced rectal cancer (LARC). Here we present the first requisite interim analysis. Methods: This is a prospective, randomized, open-label, multicenter, phase II investigator- initiated trial (IIT). Key eligibility criteria are patients with LARC and the medical need for a neoadjuvant CRT, without metastatic disease that is considered incurable by local therapies. In total 80 patients will be randomized (ratio 30:50) to receive either neoadjuvant CRT alone (50 Gy in 2 Gy fractions over 25 working days + concurrent capecitabine 1650 mg/m2/d PO) or in combination with a single dose of IPI 1 mg/kg IV at day 7, following 3 cycles of NIVO 3 mg/kg IV Q2W, starting on day 14. Patients undergo surgery within 10-12 weeks post CRT. The primary endpoint is safety of neoadjuvant CRT with sequential IPI and NIVO following surgical resection. Surgical complications are graded by the “Clavien-Dindo Classification” v2.0 and treatment-related adverse events (TRAEs) by the Common Terminology Criteria of Adverse Events (CTCAE) v5.0. Interim analyses for the surgical complication “reoperation” will be assessed after every 10th patient in the IPI/NIVO treatment arm. Reoperation numbers are compared to historically known and published ratios. If the observed case numbers are above the calculated upper 95% confidence interval (95% CIup), the study will be terminated. Results: From 06/2020-02/2022, 36 patients have been accrued, of whom 23 were randomized to the CRT+IPI/NIVO arm. Of these, the first 10 patients who underwent successful surgery were used for the present interim analysis. No patient experienced a surgical complication with the need for a reoperation (3Grade IIIb). Any surgical complication occurred in 8 (80%) patients, with the most common being Grade I (70%) and Grade II (50%) events. Conclusions: The addition of sequentially applied IPI and NIVO to neoadjuvant CRT does not increase the number of surgical reoperation rates. The study meets it’s interim analysis criteria to be safe to continue accrual. Clinical trial information: NCT04124601.

Paediatric heart transplantation recipients suffer an increased incidence of infectious, autoimmune and allergic problems. The relative roles of thymus excision and immunosuppressive treatments in contributing to these sequelae are not clear. We compared the immunological phenotypes of 25 heart transplant recipients (Tx), 10 children who underwent thymus excision during non-transplantation cardiac surgery (TE) and 25 age range-matched controls, in two age bands: 1-9 and 10-16 years. Significant differences from controls were seen mainly in the younger age band with Tx showing lower CD3 and CD4 cell counts whilst TE showed lower CD8 cell counts. Naïve T cell and recent thymic emigrant proportions and counts were significantly lower than controls in both groups in the lower age band. T cell recombination excision circle (TREC) levels were lower than controls in both groups in both age bands. There were no differences in regulatory T cells, but in those undergoing thymus excision in infancy, their proportions were higher in TE than Tx, a possible direct effect of immunosuppression. T cell receptor V beta spectratyping showed fewer peaks in both groups than in controls (predominantly in the older age band). Thymus excision in infancy was associated with lower CD8 cell counts and higher proportions of Tregs in TE compared to Tx. These data are consistent with thymus excision, particularly in infancy, being the most important influence on immunological phenotype after heart transplantation.

The search for a suitable material to promote regeneration after long‐distance peripheral nerve defects turned the spotlight on spider silk. Nerve conduits enriched with native spider silk fibers as internal guiding structures previously demonstrated a regenerative outcome similar to autologous nerve grafts in animal studies. Nevertheless, spider silk is a natural material with associated limitations for clinical use. A promising alternative is the production of recombinant silk fibers that should mimic the outstanding properties of their native counterpart. However, in vitro data on the regenerative features that native silk fibers provide for cells involved in nerve regeneration are scarce. Thus, there is a lack of reference parameters to evaluate whether recombinant silk fiber candidates will be eligible for nerve repair in vivo. To gain insight into the regenerative effect of native spider silk, our study aims to define the behavioral response of primary Schwann cells (SCs), nerve‐associated fibroblasts (FBs), and dorsal root ganglion (DRG) neurons cultured on native dragline silk from the genus Nephila and on laminin coated dishes. The established multi‐color immunostaining panels together with confocal microscopy and live cell imaging enabled the analysis of cell identity, morphology, proliferation, and migration on both substrates in detail. Our findings demonstrated that native spider silk rivals laminin coating as it allowed attachment and proliferation and supported the characteristic behavior of all tested cell types. Axonal out‐growth of DRG neurons occurred along longitudinally aligned SCs that formed sustained bundled structures resembling Bungner bands present in regenerating nerves. The migration of SCs along the silk fibers achieved the reported distance of regenerating axons of about 1 mm per day, but lacked directionality. Furthermore, rFBs significantly reduced the velocity of rSCs in co‐cultures on silk fibers. In summary, this study (a) reveals features recombinant silk must possess and what modifications or combinations could be useful for enhanced nerve repair and (b) provides assays to evaluate the regenerative performance of silk fibers in vitro before being applied as internal guiding structure in nerve conduits in vivo.

Background Stent thrombosis (ST) is a severe complication after primary percutaneous coronary intervention (pPCI) and associated with significant morbidity and mortality. Apart from procedure- and lesion-related parameters and patient-related factors. However, the underlying molecular and cellular mechanisms of ST are still not fully understood. Purpose We aimed to perform in-depth proteomic analysis of ST to understand its pathogenesis. Methods We recruited 77 patients suffering from ST after pPCI for myocardial infarction (MI). As controls, we included matched patients suffering from native vessel acute myocardial infarction (NT, n=154). Five cases of acute ST (within 24 h) and six cases of NT thrombi aspirated from the culprit site were subjected to shotgun proteomic analysis. Gene-set analysis was employed to screen for pathways differing between ST and NT. All-cause mortality was assessed using Kaplan-Meier analysis. Results 9 patients presented with acute ST (1 year, 50.6%). ST was associated with increased all-cause mortality compared to NT (mean survival 129 vs. 109 months, log-rank p=0.032). We identified a total of 2438 proteins to be expressed in both ST and NT thrombi. Gene set analysis revealed the complement system to be highly active in acute ST compared to NT. Specifically, we found factors of both the classical (complement factor [C]1q, C1s) and alternative pathway (complement factor B) to be increased in ST, along with higher levels of C2, C3, C4a, C4b, C5, C8a and C9. Conclusion This hypothesis-generating study highlights a crucial role of the complement system in the pathogenesis of acute ST. Further studies are required to validate these findings in a larger cohort. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund

Background Immune checkpoint inhibitors (ICI), such as ipilimumab (anti-cytotoxic T-lymphocyte-associated protein 4; CTLA-4) or nivolumab (anti-programmed cell death protein 1; PD-1) have been proven to be an effective strategy in solid cancers. Nevertheless, ICI seem not to be effective in microsatellite stable (MSS) tumors, as those potentially lack an immunogenic priming. Radiotherapy is capable to induce an immunogenic cell death (ICD) and subsequently an immunogenic tumor immune microenvironment (TIME). Thus, the pro-inflammatory effect of radiotherapy might restore the susceptibility of MSS tumors to ICI, leading to more pronounced tumor shrinkage, as well as to an effective anti-tumor immune response. Material and Methods In total 80 patients with a locally advanced rectal cancer (LARC) will be randomly assigned in a 30:50 ratio, to receive either standard of care (SOC) neoadjuvant chemoradiotherapy alone (CRT; 50 Gy in 2 Gy fractions with capecitabine 1650 mg/m2/d over 25 working days) or concomitant with a single dose of ipilimumab 1 mg/kg on day 7 following sequentially 3 cycles of nivolumab 3 mg/kg Q2W starting on day 14. Patients will undergo surgery within 10 to 12 weeks post CRT. The primary endpoint is safety, tolerability and feasibility assessed by the latest Clavien-Dindo classification of surgical complications and the common terminology criteria of adverse events (CTCAE). Results ClinicalTrials. gov identifier: NCT04124601. Serial liquid (plasma, serum, peripheral blood mononuclear cells) and tissue biopsies will be taken sequentially before, during and after neoadjuvant therapy. Secondary objectives are radiographic (mrTRG) and pathological (TRG) therapy response. Immune cell infiltrate of resected specimen, as well as genomic, transcriptomic, epigenomic and proteomic pattern of sequential liquid and tissue biopsies will be correlated with therapy response and clinical outcome. Conclusion This is the first in human study, which uses neoadjuvant CRT in LARC patients with concomitant ipilimumab and nivolumab, applied in a sequential approach. A detailed understanding of therapy induced changes during neoadjuvant CRT with concomitant ICI in a human translation setting will allow the application of radiotherapy as a part of novel immunotherapeutic concept. This is an investigator-initiated trial (IIT), which received a research grant and the study medications from Bristol-Myers Squibb (BMS). Disclosure Information J. Laengle: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; IIT, Research Grant & Study Medication from BMS. I. Kuehrer: None. D. Pils: None. J. Kabiljo: None. K. Woran: None. J. Stift: None. F. Herbst: None. B. Dauser: None. M. Monschein: None. P. Razek: None. S. Haegele: None. M. Herac: None. W. Hulla: None. C. Bitterman: None. F. Laengle: None. D. Tamandl: None. J. Widder: None. R. Schmid: None. M. Bergmann: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; IIT, Research Grant & Study Medication from BMS.

It is still a question of debate whether neutrophils, often found in the tumor microenvironment, mediate tumor-promoting or rather tumor-inhibiting activities. The present study focuses on the involvement of neutrophils in high grade serous ovarian cancer (HGSOC). Macroscopic features classify two types of peritoneal tumor spread in HGSOC. Widespread and millet sized lesions characterize the miliary type, while non-miliary metastases are larger and associated with better prognosis. Multi-omics and FACS data were generated from ascites samples. Integrated data analysis demonstrates a significant increase of neutrophil extracellular trap (NET)-associated molecules in non-miliary ascites samples. A co-association network analysis performed with the ascites data further revealed a striking correlation between NETosis-associated metabolites and several eicosanoids. The congruence of data generated from primary neutrophils with ascites analyses indicates the predominance of NADPH oxidase 2 (NOX)-independent NETosis. NETosis is associated with protein S100A8/A9 release. An increase of the S100A8/CRP abundance ratio was found to correlate with favorable survival of HGSOC patients. The analysis of additional five independent proteome studies with regard to S100A8/CRP ratios confirmed this observation. In conclusion, NET formation seems to relate with better cancer patient outcome.

Background: T cell density in colorectal cancer (CRC) has proven to be of high prognostic importance. Here, we evaluated the influence of a hyperfractionated preoperative short-term radiation protocol (25 Gy) on immune cell density in tumor samples of rectal cancer (RC) patients and on patient survival. In addition, we assessed spatial tumor heterogeneity by comparison of analogue T cell quantification on full tissue sections with digital T cell quantification on a virtually established tissue microarray (TMA). Methods: A total of 75 RC patients (60 irradiated, 15 treatment-na&iuml, ve) were defined for retrospective analysis. RC samples were processed for immunohistochemistry (CD3, CD8, PD-1, PD-L1). Analogue (score 0&ndash, 3) as well as digital quantification (TMA: 2 cores vs. 6 cores, mean T cell count) of marker expression in 2 areas (central tumor, CT, invasive margin, IM) was performed. Survival was estimated on the basis of analogue as well as digital marker densities calculated from 2 cores (Immunoscore: CD3/CD8 ratio) and 6 cores per tumor area. Results: Irradiated RC samples showed a significant decrease in CD3 and CD8 positive T cells, independent of quantification mode. T cell densities of 6 virtual cores approximated to T cell densities of full tissue sections, independent of individual core density or location. Survival analysis based on full tissue section quantification demonstrated that CD3 and CD8 positive T cells as well as PD-1 positive tumor infiltrating leucocytes (TILs) in the CT and the IM had a significant impact on disease-free survival (DFS) as well as overall survival (OS). In addition, CD3 and CD8 positive T cells as well as PD-1 positive TILs in the IM proved as independent prognostic factors for DFS and OS, in the CT, PD-1 positive TILs predicted DFS and CD3 and CD8 positive T cells as well as PD-1 positive TILs predicted OS. Survival analysis based on virtual TMA showed no impact on DFS or OS. Conclusion: Spatial tumor heterogeneity might result in inadequate quantification of immune marker expression, however, if using a TMA, 6 cores per tumor area and patient sample represent comparable amounts of T cell densities to those quantified on full tissue sections. Consistently, the tissue area used for immune marker quantification represents a crucial factor for the evaluation of prognostic and predictive biomarker potential.

Background: Pulmonary metastasectomy is one of the cornerstones in the treatment of oligometastatic colorectal cancer (CRC). However, the selection of patients who benefit from a surgical resection is difficult. Mutational profiling has become an essential part of diagnosis and treatment of malignant disease. Despite this, comprehensive data on the mutational profile of CRC and its clinical impact in the context of pulmonary metastasectomy is sparse. We therefore aimed to provide a complete mutational status of CRC pulmonary metastases (PM) and corresponding primary tumors by targeted next-generation sequencing (tNGS), and correlate sequencing data with clinical outcome variables. Methods: Case-matched, formalin-fixed paraffin embedded surgical specimens of lung metastases (n=47) and matched primary CRC (n=24) were sequenced using the TruSeq Amplicon Cancer Panel (Illumina platform). Penalized Cox regression models were applied to identify mutations with prognostic impact. Results: Mutations were found most frequently in APC, TP53 and KRAS , in both PM and matched primary tumors. Concordance between primary tumors and PM was 83.5%. Adaptive elastic-net regularized Cox regression models identified mutations being prognostic for time to pulmonary recurrence ( EGFR, GNAQ, KIT, MET , and PTPN11 ) and for overall survival (OS) ( PDGFRA, SMARCB1 , and TP53 ). Conclusions: Our findings suggest that CRC PM harbor a variety of conserved and de novo mutations. We could identify a mutational profile predicting clinical outcome after pulmonary metastasectomy. Moreover, our data provide a rationale for future targeted therapies of patients with CRC lung metastases.

Research question As microRNA (miRNA) are stable in circulation, this study tested whether they could serve as putative non-invasive biomarkers for endometriosis, and their expression differences between endometriosis patients and controls. It also addressed whether the combination of differently expressed miRNA together with clinical parameters in a statistical model could distinguish between endometriosis patients and controls. Design This prospective cohort study explored the possibility of using changes in extracellular miRNA spectra in plasma of 51 patients with endometriosis compared with 41 controls combined with clinical data as non-invasive biomarkers for the disease. The project was divided into three different phases for biomarker screening, discovery and validation. The differences in expression levels of plasma miRNA obtained from women with and without endometriosis were analysed with quantitative PCR-based microarrays. The diagnostic performance of the selected individual and/or combined differentially expressed miRNA candidates and clinical parameters was assessed using in silico bioinformatics modelling and receiver operating characteristic curve analysis. Results Data showed that a specific plasma miRNA signature is associated with endometriosis and that hsa-miR-154-5p, which alone or in combination with hsa-miR-196b-5p, hsa-miR-378a-3p, and hsa-miR-33a-5p and the clinical parameters of body mass index and age, are potentially applicable for non-invasive diagnosis of the disease. Changes in the levels of expression of certain circulating plasma miRNA also occurred within the phases of the menstrual cycle. Conclusions miRNA seem to be promising candidates for the non-invasive diagnosis of endometriosis. Further, other clinical parameters may help in distinguishing women suffering from endometriosis from healthy individuals.

The transcriptome needs to be tightly regulated by mechanisms that include transcription factors, enhancers, and repressors as well as non‐coding RNAs. Besides this dynamic regulation, a large part of phenotypic variability of eukaryotes is expressed through changes in gene transcription caused by genetic variation. In this study, we evaluate genome‐wide structural genomic variants (SVs) and their association with gene expression in the human heart. We detected 3,898 individual SVs affecting all classes of gene transcripts (e.g., mRNA, miRNA, lncRNA) and regulatory genomic regions (e.g., enhancer or TFBS). In a cohort of patients (n = 50) with dilated cardiomyopathy (DCM), 80,635 non‐protein‐coding elements of the genome are deleted or duplicated by SVs, containing 3,758 long non‐coding RNAs and 1,756 protein‐coding transcripts. 65.3% of the SV‐eQTLs do not harbor a significant SNV‐eQTL, and for the regions with both classes of association, we find similar effect sizes. In case of deleted protein‐coding exons, we find downregulation of the associated transcripts, duplication events, however, do not show significant changes over all events. In summary, we are first to describe the genomic variability associated with SVs in heart failure due to DCM and dissect their impact on the transcriptome. Overall, SVs explain up to 7.5% of the variation of cardiac gene expression, underlining the importance to study human myocardial gene expression in the context of the individual genome. This has immediate implications for studies on basic mechanisms of cardiac maladaptation, biomarkers, and (gene) therapeutic studies alike.

Background: Biochemical DNA modification resembles a crucial regulatory layer among genetic information, environmental factors, and the transcriptome. To identify epigenetic susceptibility regions and novel biomarkers linked to myocardial dysfunction and heart failure, we performed the first multi-omics study in myocardial tissue and blood of patients with dilated cardiomyopathy and controls. Methods: Infinium human methylation 450 was used for high-density epigenome-wide mapping of DNA methylation in left-ventricular biopsies and whole peripheral blood of living probands. RNA deep sequencing was performed on the same samples in parallel. Whole-genome sequencing of all patients allowed exclusion of promiscuous genotype-induced methylation calls. Results: In the screening stage, we detected 59 epigenetic loci that are significantly associated with dilated cardiomyopathy (false discovery corrected P ≤0.05), with 3 of them reaching epigenome-wide significance at P ≤5×10 −8 . Twenty-seven (46%) of these loci could be replicated in independent cohorts, underlining the role of epigenetic regulation of key cardiac transcription regulators. Using a staged multi-omics study design, we link a subset of 517 epigenetic loci with dilated cardiomyopathy and cardiac gene expression. Furthermore, we identified distinct epigenetic methylation patterns that are conserved across tissues, rendering these CpGs novel epigenetic biomarkers for heart failure. Conclusions: The present study provides to our knowledge the first epigenome-wide association study in living patients with heart failure using a multi-omics approach.

Purpose: Cancer metabolism is characterized by alterations including aerobic glycolysis, oxidative phosphorylation, and need of fuels and building blocks. Experimental Design: Targeted metabolomics of preoperative and follow-up sera, ascites, and tumor tissues, RNA sequencing of isolated tumor cells, local and systemic chemokine, and local immune cell infiltration data from up to 65 high-grade serous ovarian cancer patients and 62 healthy controls were correlated to overall survival and integrated in a Systems Medicine manner. Results: Forty-three mainly (poly)unsaturated glycerophospholipids and four essential amino acids (citrulline) were significantly reduced in patients with short compared with long survival and healthy controls. The glycerophospholipid fingerprint is identical to the fingerprint from isolated (very) low-density lipoproteins (vLDL), indicating that the source of glycerophospholipids consumed by tumors is (v)LDL. A glycerophospholipid-score (HR, 0.46; P = 0.007) and a 100-gene signature (HR, 0.65; P = 0.004) confirmed the independent impact on survival in training (n = 65) and validation (n = 165) cohorts. High concentrations of LDLs and glycerophospholipids were independently predictors for favorable survival. Patients with low glycerophospholipids presented with more systemic inflammation (C-reactive protein and fibrinogen negatively and albumin positively correlated) but less adaptive immune cell tumor infiltration (lower tumor and immune cell PD-L1 expression), less oxygenic respiration and increased triglyceride biosynthesis in tumor cells, and lower histone expressions, correlating with higher numbers of expressed genes and more transcriptional noise, a putative neo-pluripotent tumor cell phenotype. Conclusions: Low serum phospholipids and essential amino acids are correlated with worse outcome in ovarian cancer, accompanied by a specific tumor cell phenotype. Clin Cancer Res; 23(8); 2081–92. ©2016 AACR.

It is still a question of debate whether neutrophils, often found in the tumor microenvironment, mediate tumor-promoting or rather tumor-inhibiting activities. The present study focusses on the involvement of neutrophils in high grade serous ovarian cancer (HGSOC). Multi-omics data comprising proteomics, eicosadomics, metabolomics, Luminex-based cytokinomics, and FACS data were generated from ascites samples. Integrated data analysis demonstrates a significant increase of neutrophil extracellular trap-(NET) associated molecules in non-miliary ascites samples. A co-association network analysis performed with the ascites data further revealed a striking co-correlation between NETosis-associated metabolites with several eicosanoids. Investigating primary neutrophils from healthy domors, NET formation was induced using ionomycin or phorbol ester. Data congruence with ascites analyses indicated the predominance of NOX-independent NETosis. NETosis is associated with S100A8/A9 release. An increase of the S100A8/CRP abundance ratio was found to correlate with improved survival of HGSOC patients. The analysis of additional five independent proteome studies with regard to S100A8/CRP ratios confirmed this observation. In conclusion, here we present evidence that increased NET formation relates to improved outcomes in cancer patients.Graphical abstractNETs releasing neutrophils through detaching of small tumor nods dictate the building of bigger in size and fewer in number of tumors in the non-miliary spreading tumor. Increased angiogenesis associated with increased blood circulation may contribute to less suppressive effects on NETs formation in the non-miliary tumor type. Tumor origin,i.e. fallopian tube for the miliary or ovary for the non-miliary tumors, may influence the angiogenesis and therewith – through facilitating of neutrophils activation – (co)determine the type of tumor spread.

TPS3623 Background: Immune checkpoint inhibitors (ICI), such as ipilimumab (anti-cytotoxic T-lymphocyte-associated protein 4) or nivolumab (anti-programmed cell death protein 1) have been proven to be an effective strategy in solid cancers. However, ICI seem not to be effective in microsatellite stable (MSS) cancers. As they might lack an immunogenic priming, radiotherapy (RT) is capable to induce an immunogenic cell death (ICD) and subsequently an immunogenic tumor immune microenvironment (TIME). Thus, RT might restore the susceptibility of MSS tumors to ICI and consequently leading to an effective anti-tumor immune response. Methods: This is a prospective, randomized, open-label, multicenter, phase II investigator-initiated clinical trial (IIT), including patients with locally advanced rectal cancer (LARC). Patients receive either neoadjuvant chemoradiotherapy (CRT) alone (50 Gy in 2 Gy fractions over 25 working days + capecitabine 1650 mg/m2/d PO) or in combination with ipilimumab (1 mg/kg IV on day 7) and nivolumab (3 mg/kg IV on day 14, 28 and 42). Patients will undergo surgery within 10-12 weeks post CRT. The primary endpoint is incidence of treatment-emergent adverse events (AEs) assessed by the Clavien-Dindo classification of surgical complications and the common terminology criteria of adverse events (CTCAE). Secondary objectives are radiographic and pathological therapy response. Serial liquid (plasma, serum and peripheral blood mononuclear cells) and tissue biopsies will be taken before, during and after neoadjuvant treatment. Genomic, transcriptomic, epigenomic and proteomic pattern of liquid and tissue biopsies, as well as the immune cell infiltrate of resected specimen, will be correlated with therapy response and clinical outcome. Currently 8 of planned 80 patients have been enrolled. Registration numbers: NCT no. NCT04124601, EudraCT no. 2019-003865-17. Clinical trial information: NCT04124601.