Background Magnetic resonance (MR) diffusion tensor imaging (DTI) is a new, non-invasive method to investigate arterial vessel walls. High resolution diffusion weighted imaging (DWI) in combination with a two-dimensional (2D) diffusion gradient sampling scheme has recently been demonstrated as feasible for evaluating diffusivity in the vessel wall of human carotids. We aimed to identify associations of altered diffusivity in human carotids with presence or absence of cardiovascular disease, body mass index (BMI), sex and age in a clinical setting. Methods In this single center case-control study we used DWI in combination with a 2D gradient in a 3 T MRI scanner to evaluate diffusivity parameters in carotid vessel walls of clinical patients with known cardiovascular disease (n = 17) and healthy controls (n = 21). We used a read-out segmented EPI (rs-EPI) sequence for DWI. Results A group comparison showed significantly decreased fractional anisotropy (FA) in patients older than 60 years (p < 0.001) and in patients with a BMI higher than 25 (p = 0.019). Patients with cardiovascular disease had higher values for mean diffusivity (p = 0.005) than patients without cardiovascular disease. A multiple linear regression analysis showed age and sex to be associated with FA. Conclusion Two-dimensional vessel wall DTI of human carotids is feasible for clinical research. Besides patient age, we identified Sex, BMI, or the presence of cardiovascular disease as relevant factors for carotid vessel wall diffusivity. Decreased FA values might indicate early-stage atherosclerosis and vascular ageing.

Lipoprotein(a) (Lp(a)) is considered an independent risk factor for cardiovascular diseases. The plasma concentration of Lp(a) is largely genetically determined but varies over a wide range within the population. This study investigated changes in Lp(a) levels after an acute myocardial infarction. Patients who underwent coronary angiography due to an ST elevation myocardial infarction were enrolled (n = 86), and Lp(a) levels were measured immediately after the intervention, one day, two days, and at a post-discharge follow-up visit at 3 to 6 months after the acute myocardial infarction. Median Lp(a) levels increased from a median of 7.9 mg/dL (3.8–37.1) at hospital admission to 8.4 mg/dL (3.9–35.4) on the following day, then to 9.3 mg/dL (3.7–39.1) on day two (p < 0.001), and to 11.2 mg/dL (4.4–59.6) at the post-discharge follow-up (p < 0.001). Lp(a) levels were the lowest during the acute myocardial infarction and started to increase significantly immediately thereafter, with the highest levels at the post-discharge follow-up. The moderate but significant increase in Lp(a) in people with acute myocardial infarction appears to be clinically relevant on an individual basis, especially when specific Lp(a) cut-off levels are supposed to determine the initiation of future treatment. Hence, a repeated measurement of Lp(a) after myocardial infarction should be performed.

Background: PCSK9 antibodies strongly reduce LDL cholesterol. The effects of PCSK9 antibodies on triglyceride metabolism are less pronounced. The present study aimed to investigate in detail the effects of alirocumab on triglycerides, triglyceride-rich lipoproteins, and lipase regulators. Methods: A total of 24 patients with an indication for treatment with PCSK9 antibodies were recruited. There were two visits at the study site: the first before initiation of treatment with alirocumab and the second after 10 weeks of treatment. Fat-tolerance tests, nuclear magnetic resonance spectroscopy, and enzyme-linked immunosorbent assays were performed to analyze lipid metabolism. Results: A total of 21 participants underwent the first and second investigation. Among these, two participants only received alirocumab twice and 19 patients completed the trial per protocol. All of them had atherosclerotic vascular disease. There was no significant effect of alirocumab treatment on fasting triglycerides, post-prandial triglycerides, or lipoprotein-lipase regulating proteins. Total, large, and small LDL particle concentrations decreased, while the HDL particle concentration increased (all p < 0.001). Mean total circulating PCSK9 markedly increased in response to alirocumab treatment (p < 0.001). Whereas PCSK9 increased more than three-fold in all 19 compliant patients, it remained unchanged in those two patients with two injections only. Conclusion: Significant effects of alirocumab on triglyceride metabolism were not detectable in the ALIROCKS trial. The total circulating PCSK9 concentration might be a useful biomarker to differentiate non-adherence from non-response to PCSK9 antibodies.

Background: Short-term effects of alirocumab on vascular function have hardly been investigated. Moreover, there is a scarce of reliable non-invasive methods to evaluate atherosclerotic changes of the vasculature. The ALIROCKS trial was performed to address these issues using standard ultrasound-based procedures and a completely novel magnetic resonance-based imaging technique. Methods: A total of 24 patients with an indication for treatment with PCSK9 antibodies were recruited. There were 2 visits to the study site, the first before initiation of treatment with alirocumab and the second after 10 weeks of treatment. The key outcome measures included the change of carotid vessel wall fractional anisotropy, a novel magnetic resonance-based measure of vascular integrity, and the changes of carotid intima-media thickness and flow-dependent dilatation of the brachial artery measured with ultrasound. Results: A total of 19 patients completed the trial, 2 patients stopped treatment, 3 patients did not undergo the second visit due to the COVID pandemic. All of them had atherosclerotic vascular disease. Their mean (standard deviation) LDL-cholesterol concentration was 154 (85) mg/dL at baseline and was reduced by 76 (44) mg/dL in response to alirocumab treatment (p < 0.001, n = 19). P-selectin and vascular endothelial growth factors remained unchanged. Flow-dependent dilatation of the brachial artery (+41%, p = 0.241, n = 18), carotid intima-media thickness (p = 0.914, n = 18), and fractional anisotropy of the carotid artery (p = 0.358, n = 13) also did not significantly change. Conclusion: Despite a nominal amelioration for flow-dependent dilatation, significant effects of short-term treatment with alirocumab on vascular function were not detectable. More work would be needed to evaluate, whether fractional anisotropy may be useful in clinical atherosclerosis research.

Objective Proprotein convertase subtilisin/kexin type 9 inhibition can be an effective treatment in patients with primary hypercholesterolemia, particularly in cases with concomitant coronary heart disease, peripheral artery occlusive disease or cerebrovascular occlusive disease for secondary prevention after an acute atherosclerotic ischemic event. The primary objective of the PEARL-AT study was to assess effectiveness and safety of alirocumab in a real-world setting in Austria. Methods Non-interventional, prospective study conducted across Austria between September 2016 and July 2018. 113 patients, for whom the decision for treatment with alirocumab according to the Austrian Summary of Product Characteristics (SmPC) was made, were enrolled and were followed-up over 24 weeks. The primary endpoint of the study was the average change of low density lipoprotein cholesterol (LDL-C) levels by week 24. Results In total, 112 patients with at least one post-baseline visit were included. Alirocumab was initiated using 75 mg (57.1%) and 150 mg (42.9%) every two weeks. Average LDL-C levels decreased by 75.0 mg/dl at week 24 in 87 patients with available LDL-C at baseline and week 24 (in 25 patients LDL-C was missing at least at one time point). The mean relative change of LDL-C was -50.0% (median: 57.8%, SD: 28.4). Throughout the study, 46 adverse events were documented in 21 (18.6%) patients. The most frequent adverse events were gastrointestinal disorders. Conclusions The present data indicate a good overall efficacy of alirocumab in a real-world Austrian population. Effectiveness and safety were both in line with the clinical trial program as well as previous real-world observations.

Hematologic spread of carcinoma results in incurable metastasis; yet, the basic characteristics and travel mechanisms of cancer cells in the bloodstream are unknown. We have established a fluid phase biopsy approach that identifies circulating tumor cells (CTCs) without using surface protein-based enrichment and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. This 'HD-CTC' assay finds >5 HD-CTCs mL(-1) of blood in 80% of patients with metastatic prostate cancer (n = 20), in 70% of patients with metastatic breast cancer (n = 30), in 50% of patients with metastatic pancreatic cancer (n = 18), and in 0% of normal controls (n = 15). Additionally, it finds HD-CTC clusters ranging from 2 HD-CTCs to greater than 30 HD-CTCs in the majority of these cancer patients. This initial validation of an enrichment-free assay demonstrates our ability to identify significant numbers of HD-CTCs in a majority of patients with prostate, breast and pancreatic cancers.

194 Background: We have established a fluid phase biopsy approach that identifies CTCs which preserves cytologic features in high-definition (HD) for diagnostic pathology without using immune or surface receptor-based enrichment. HD-CTCs identified with this approach can be used for enumeration and molecular characterization. Methods: Blood was collected from metastatic prostate cancer patients and normal donors in Cyto-Chex tubes (Streck, Omaha, NE) as part of IRB approved protocols at each site. Following erythrocyte lysis, 3 million nucleated cells were deposited on a glass slide. Samples were incubated with a pan-cytokeratin (CK), CD45, and androgen receptor (AR) antibodies and counter-stained with DAPI. LNCaP cells were spiked into normal blood. Images were obtained with a fluorescent scanning microscope and analyzed with a computer algorithm. Candidate HD-CTCs were subsequently verified by expert readers. Slides were re-imaged for quantitative analysis using at a fixed exposure and gain. Results: A total of 227 CTCs from ten patients were compared to 20 LNCaP cells. The median (range) HD-CTCs in this cohort was: 9 (1-62) cells/ml. The mean ± standard deviation measurements in HD-CTCs were observed: CK intensity 60.4±154; total cell area 89.0 ± 53.8 µm2; nuclear area 61.1 ± 36.0 µm2. LNCaP cells spiked into normal blood gave the following values: CK intensity 1166+/−306; total cell area 143 ± 48.1 µm2; nuclear area 63.1 ± 18.6 µm2. CTCs were additionally classified as either AR positive (AR+) or AR negative (AR−). 37 of the 227 (16.3%) HD-CTCs were AR+. The average CK intensity was significantly higher in AR+ versus AR− cells at 174.23 and 39.86, respectively (p<0.001). The AR expression intensity in AR+ HD-CTCs and LNCaP cells was comparable at 979.4 and 902.2, respectively (p=0.824). Conclusions: We find a positive association between AR and CK expression on a per cell basis. Further, we find AR is expressed at comparable levels in CTCs from patients and human prostate cancer cells in culture. The HD-CTC based approach may be used for enumeration and molecular interrogation of CTCs in patients with prostate cancer.

Cutaneous melanoma is a tumor with rising incidence and a very poor prognosis at the disseminated stage. Melanomas are characterized by frequent mutations in BRAF and also by overexpression of fibroblast growth factor 2 (FGF2), offering opportunities for therapeutic intervention. We investigated inhibition of FGF signaling and its combination with dacarbazine or BRAF inhibitors as an antitumor strategy in melanoma. The majority of melanoma cell lines displayed overexpression of FGF2 but also FGF5 and FGF18 together with different isoforms of FGF receptors (FGFRs) 1-4. Blockade of FGF signals with dominant-negative receptor constructs (dnFGFR1, 3, or 4) or small-molecule inhibitors (SU5402 and PD166866) reduced melanoma cell proliferation, colony formation, as well as anchorage-independent growth, and increased apoptosis. DnFGFR constructs also significantly inhibited tumor growth in vivo. Combination of FGF inhibitors with dacarbazine showed additive or antagonistic effects, whereas synergistic drug interaction was observed when combining FGFR inhibition with the multikinase/BRAF inhibitor sorafenib or the V600E mutant-specific BRAF inhibitor RG7204. In conclusion, FGFR inhibition has antitumor effects against melanoma cells in vitro and in vivo. Combination with BRAF inhibition offers a potential for synergistic antimelanoma effects and represents a promising therapeutic strategy against advanced melanoma.

Circulating tumor cell (CTC) counts are an established prognostic marker in metastatic prostate, breast, and colorectal cancer. However, there is little knowledge about CTC incidence rates and morphologic heterogeneity in non-small cell lung cancer (NSCLC) and their correlation to disease progression and usability for clinical staging. Here, we present a fluid phase biopsy approach that identifies CTCs without surface receptor-based enrichment, and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. The HD-CTC assay will be used to monitor HD-CTC counts and morphology in early stage, locally advanced, and metastatic NSCLC patient over time. 150 newly diagnosed NSCLC patients are being enrolled for this project. At the time of diagnosis 50 of the patients will have early stage cancers (stage I-II); 50 patients will have locally advanced (stage III) cancers, and 50 will be patients presenting with metastatic (stage IV) disease. Blood samples from each patient will be obtained at the time of diagnosis and 6 months later. We present here an interim data set that shows an incidence rate of 78% at baseline with a median CTC count of 19.5/ml of blood (range 0-441.6). Our preliminary data demonstrates that, despite stringent morphologic inclusion criteria for the definition of HD-CTCs, our HD-CTC assay shows high sensitivity in the detection of lung cancer CTCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4151. doi:10.1158/1538-7445.AM2011-4151

Activins are secreted proteins belonging to the TGF-β family of signaling molecules. Activin signals are crucial for differentiation and regulation of cell proliferation and apoptosis in multiple tissues. Signal transduction by activins relies mainly on the Smad pathway, although the importance of crosstalk with additional pathways is increasingly being recognized. Activin signals are kept in balance by antagonists at multiple levels of the signaling cascade. Among these, follistatin and FLRG, two members of the emerging family of follistatin-like proteins, can bind secreted activins with high affinity, thereby blocking their access to cell surface-anchored activin receptors. In the liver, activin A is a major negative regulator of hepatocyte proliferation and can induce apoptosis. The functions of other activins expressed by hepatocytes have yet to be more clearly defined. Deregulated expression of activins and follistatin has been implicated in hepatic diseases including inflammation, fibrosis, liver failure and primary cancer. In particular, increased follistatin levels have been found in the circulation and in the tumor tissue of patients suffering from hepatocellular carcinoma as well as in animal models of liver cancer. It has been argued that up-regulation of follistatin protects neoplastic hepatocytes from activin-mediated growth inhibition and apoptosis. The use of follistatin as biomarker for liver tumor development is impeded, however, due to the presence of elevated follistatin levels already during preceding stages of liver disease. The current article summarizes our evolving understanding of the multi-faceted activities of activins and follistatins in liver physiology and cancer.