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There is accumulating evidence that pro-inflammatory features are inherent to mitochondrial DNA and oxidized DNA species. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is the most frequently studied oxidatively generated lesion. Modified DNA reaches the circulation upon cell apoptosis, necrosis or neutrophil extracellular trap (NET) formation. Standard chromatography-based techniques for the assessment of 8-oxodGuo imply degradation of DNA to a single base level, thus precluding the attribution to a nuclear or mitochondrial origin. We therefore aimed to establish a protocol for the concomitant assessment of oxidized mitochondrial and nuclear DNA from human plasma samples. We applied immunoprecipitation (IP) for 8-oxodGuo to separate oxidized from non-oxidized DNA species and subsequent quantitative polymerase chain reaction (qPCR) to assign them to their subcellular source. The IP procedure failed when applied directly to plasma samples, i.e. isotype control precipitated similar amounts of DNA as the specific 8-oxodGuo antibody. In contrast, DNA isolation from plasma prior to the IP process provided assay specificity with little impact on DNA oxidation status. We further optimized sensitivity and efficiency of qPCR analysis by reducing amplicon length and targeting repetitive nuclear DNA elements. When the established protocol was applied to plasma samples of abdominal aortic aneurysm (AAA) patients and control subjects, the AAA cohort displayed significantly elevated circulating non-oxidized and total nuclear DNA and a trend for increased levels of oxidized mitochondrial DNA. An enrichment of mitochondrial versus nuclear DNA within the oxidized DNA fraction was seen for AAA patients. Regarding the potential source of circulating DNA, we observed a significant correlation of markers of neutrophil activation and NET formation with nuclear DNA, independent of oxidation status. Thus, the established method provides a tool to detect and distinguish the release of oxidized nuclear and mitochondrial DNA in human plasma and offers a refined biomarker to monitor disease conditions of pro-inflammatory cell and tissue destruction., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Monitoring of abdominal aortic aneurysms (AAAs) is currently based on serial measurements of maximum aortic diameter. Additional assessment of aneurysm volume has previously been proposed to possibly improve growth prediction and treatment decisions. To evaluate the use of supplementing volume measurements, the authors aimed to characterise the growth distribution of AAA volume and to compare the growth rates of the maximum diameter and volume at the patient level., Methods: Maximum diameter and volume were monitored every 6 months in 84 patients with small AAAs, with a total of 331 computed tomographic angiographies (with initial maximum diameters of 30-68 mm). A previously developed statistical growth model for AAAs was applied to assess the growth distribution of volume and to compare individual growth rates for volume and for maximum diameter., Results: The median (25-75% quantile) expansion in volume was 13.4 (6.5-24.7) % per year. Cube root transformed volume and maximum diameter showed a closely linear association with a within-subject correlation of 0.77. At the surgery threshold maximum diameter of 55 mm, the median (25-75% quantile) volume was 132 (103-167) ml. In 39% of subjects, growth rates for volume and maximum diameter were equivalent, in 33% growth was faster in volume and in 27% growth was faster in maximum diameter., Conclusion: At the population level, volume and maximum diameter show a substantial association such that the average volume is approximately proportional to the average maximum diameter raised to a power of three. At the individual level, however, in the majority of patient's AAAs grow at different pace in different dimensions. Hence, closer monitoring of aneurysms with sub-critical diameter but suspicious morphology may benefit from complementing maximum diameter by volume or related measurements., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Objective: This retrospective study evaluates the performance of UK National Institute for Health and Care Excellence (NICE) Guidelines on management of ruptured abdominal aortic aneurysms in a "real world setting" by emulating a hypothetical target trial with data from two European Aortic Centers., Methods: Clinical data was retrospectively collected for all patients who had undergone ruptured endovascular aneurysm repair (rEVAR) and ruptured open surgical repair (rOSR). Survival analysis was performed comparing NICE compliance to usual care strategy. NICE compliers were defined as: female patients undergoing rEVAR; male patients >70 years old undergoing rEVAR; and male patients ≤70 years old undergoing rOSR. Hemodynamic instability was considered additionally., Results: This multicenter study included 298 patients treated for rAAA. The majority of patients were treated with rOSR (186 rOSR vs. 112 rEVAR). Overall, 184 deaths (68 [37%] with rEVAR and 116 [63%] with rOSR) were observed during the study period. Overall survival under usual care was 69.2% at 30 days, 56.5% at one year, and 42.4% at 5 years. NICE compliance gave survival outcomes of 73.1% at 30 days, 60.2% at 1 year and 42.9% at 5 years. The risk ratios at these time points, comparing NICE-compliance to usual care, were 0.88, 0.92 and 0.99, respectively., Conclusions: We support NICE recommendations to manage men below the age of 71 years and hemodynamic stability with rOSR. There was a slight survival advantage for NICE compliers overall, in men >70 years and women of all ages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Eilenberg, Waduud, Davies, Bailey, Scott, Wolf, Sotir, Lakowitsch, Kaider, Heinze, Brostjan, Domenig and Neumayer.)

Complex endovascular aortic repair (coEVAR) of thoracoabdominal aortic aneurysms (TAAA) has greatly evolved in the past decades. Despite substantial improvements of postoperative care, spinal cord injury (SCI) remains the most devastating complication of coEVAR being associated with impaired patient outcome and having an impact on long-term survival. The rising number of challenges of coEVAR, essentially associated with an extensive coverage of critical blood vessels supplying the spinal cord, resulted in the implementation of dedicated SCI prevention protocols. In addition to maintenance of adequate spinal cord perfusion pressure (SCPP), early detection of SCI plays an integral role in intra- and postoperative patient care. However, this is challenging due to difficulties with clinical neurological examinations during patient sedation in the postoperative setting. There is a rising amount of evidence, suggesting that subclinical forms of SCI might be accompanied by an elevation of biochemical markers, specific to neuronal tissue damage. Addressing this hypothesis, several studies have attempted to assess the potential of selected biomarkers with regard to early SCI diagnosis. In this review, we discuss biomarkers measured in patients undergoing coEVAR. Once validated in future prospective clinical studies, biomarkers of neuronal tissue damage may potentially add to the armamentarium of modalities for early SCI diagnosis and risk stratification.

Background: Abdominal aortic aneurysm (AAA) is a multifactorial vascular disease associated with high morbidity and mortality. Currently, surgical intervention is the only treatment option, and there is no drug therapy available for AAA. Hence, surveillance of AAA until indication for surgery may impact patient quality of life (QoL). There is a paucity of high-quality observational data on health status and QoL, particularly among AAA patients participating in randomized controlled trials. The objective of this study was to compare the QoL scores of AAA patients on surveillance to those of AAA patients enrolled in the MetAAA trial., Material and Methods: Overall, 54 MetAAA trial patients and 23 AAA patients under regular surveillance for small AAA (part of a longitudinal monitoring study) were asked to complete three established and validated (in total 561 longitudinally collected) QoL questionnaires: the 36-Item Short Form Health Survey (SF-36), the Aneurysm Symptom Rating Questionnaire (ASRQ), and the Aneurysm-Dependent Quality of Life questionnaire (ADQoL)., Results: A superior health status and QoL was found in AAA patients participating in the MetAAA trial compared to AAA patients under regular surveillance. In detail, MetAAA trial patients showed superior general health perception ( P =0.012), higher energy level ( P =0.036) as well as enhanced emotional well-being ( P =0.044) and fewer limitations due to general malaise ( P =0.021), which was subsequently reflected in an overall superior current QoL score ( P =0.039) compared to AAA patients under regular surveillance., Conclusion: AAA patients enrolled in the MetAAA trial showed superior health status and QoL compared to AAA patients under regular surveillance., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Patients with cancer have an increased risk of developing venous thromboembolism. Neutrophils and neutrophil extracellular traps (NETs) reportedly influence the risk of cancer-associated thrombosis. Subpopulations of high and low-density neutrophils (HDN/LDN) are of specific interest, as they might have different functions in cancer patients., Objectives: We aimed to investigate differences between HDNs and LDNs of patients with lung cancer and healthy controls, and their ability of activation and NET formation., Methods: Within the framework of the Vienna Cancer and Thrombosis Study, a prospective observational cohort study, HDNs and LDNs from 20 patients with lung cancer and 20 controls were isolated by density gradient centrifugation. The ability of neutrophil subpopulations for activation and NET formation was investigated by flow cytometry., Results: Compared to controls, patients with cancer had higher numbers of total leukocytes, HDNs, and LDNs. LDNs of patients were more frequently in an activated state (CD62L↓/CD16↑) at baseline (median [IQR] 5.9% [3.4-8.8] vs 2.5% [1.6-6.7]). HDNs and LDNs from patients showed a significantly increased response to stimulation with ionomycin (CD11b HDN: 98.5 [95.4-99.4] vs 41.7 [13.4-91.6]; LDN: 82.9 [63-94] vs 39.6 [17.3-72.1]). In addition, HDNs from patients showed a higher capability of NET formation after ionomycin stimulation compared to HDNs from healthy controls (18509.5 [12242.5-29470.3] vs 10001 [6618.8-18384.3])., Conclusion: Protumorigenic LDNs were elevated, and neutrophil subpopulations showed an increased activation profile and ability for NET formation in patients with cancer. These mechanisms might be involved in tumor promotion and contribute to the prothrombotic phenotype of neutrophils in cancer., (© Bsc1 |.)

Interleukin-4 (IL-4) and its receptors (IL-4R) promote the proliferation and polarization of macrophages. However, it is unknown if IL-4R also influences monocyte homeostasis and if steady state IL-4 levels are sufficient to affect monocytes. Employing full IL-4 receptor alpha knockout mice (IL-4Rα -/- ) and mice with a myeloid-specific deletion of IL-4Rα (IL-4Rα f/f LysM cre ), we show that IL-4 acts as a homeostatic factor regulating circulating monocyte numbers. In the absence of IL-4Rα, murine monocytes in blood were reduced by 50% without altering monocytopoiesis in the bone marrow. This reduction was accompanied by a decrease in monocyte-derived inflammatory cytokines in the plasma. RNA sequencing analysis and immunohistochemical staining of splenic monocytes revealed changes in mRNA and protein levels of anti-apoptotic factors including BIRC6 in IL-4Rα -/- knockout animals. Furthermore, assessment of monocyte lifespan in vivo measuring BrdU + cells revealed that the lifespan of circulating monocytes was reduced by 55% in IL-4Rα -/- mice, whereas subcutaneously applied IL-4 prolonged it by 75%. Treatment of human monocytes with IL-4 reduced the amount of dying monocytes in vitro. Furthermore, IL-4 stimulation reduced the phosphorylation of proteins involved in the apoptosis pathway, including the phosphorylation of the NFκBp65 protein. In a cohort of human patients, serum IL-4 levels were significantly associated with monocyte counts. In a sterile peritonitis model, reduced monocyte counts resulted in an attenuated recruitment of monocytes upon inflammatory stimulation in IL-4Rα f/f LysM cre mice without changes in overall migratory function. Thus, we identified a homeostatic role of IL-4Rα in regulating the lifespan of monocytes in vivo., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The maximal aortic diameter is the only clinically applied predictor of abdominal aortic aneurysm (AAA) progression and indicator for surgical repair. Circulating biomarkers resulting from AAA pathogenesis are attractive candidates for the diagnosis and prognosis of aneurysmal disease. Due to the reported role of interleukin 33 in AAA development, we investigated the corresponding circulating receptor molecules of soluble suppression of tumorigenesis 2 (sST2) in AAA patients regarding their marker potential in diagnosis and prognosis. We conducted a single-center retrospective cohort study in a diagnostic setting, measuring the circulating serum sST2 protein levels of 47 AAA patients under surveillance, matched with 25 peripheral artery disease (PAD) patients and 25 healthy controls. In a prognostic setting, we analyzed the longitudinal monitoring data of 50 monitored AAA patients. Slow versus fast AAA progression was defined as a <2 or ≥2 mm increase in AAA diameter over 6 months and a <4 or ≥4 mm increase over 12 months. Additionally, the association of circulating serum sST2 and AAA growth was investigated using a specifically tailored log-linear mixed model. Serum sST2 concentrations were significantly increased in AAA patients compared with healthy individuals: the median of AAA patient cohort was 112.72 ng/mL (p = 0.025) and that of AAA patient cohort 2 was 14.32 ng/mL (p = 0.039) versus healthy controls (8.82 ng/mL). Likewise, PAD patients showed significantly elevated sST2 protein levels compared with healthy controls (the median was 12.10 ng/mL; p = 0.048) but similar concentrations to AAA patients. Additionally, sST2 protein levels were found to be unsuited to identifying fast AAA progression over short-term periods of 6 or 12 months, which was confirmed by a log-linear mixed model. In conclusion, the significantly elevated protein levels of sST2 detected in patients with vascular disease may be useful in the early diagnosis of AAA but cannot distinguish between AAA and PAD or predict AAA progression.

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse models, are applied to advance the understanding of the disease pathogenesis and to identify potential therapeutic targets. Testing novel drug candidates to block AAA growth in these models generally requires repeated drug administration during the time course of the experiment. Here, we describe a compiled protocol for AAA induction, insertion of an intravenous catheter to facilitate prolonged therapy, and serial AAA monitoring by 3D ultrasound. Aneurysms are induced in apolipoprotein E (ApoE) deficient mice by angiotensin II release over 28 days from osmotic mini-pumps implanted subcutaneously into the mouse back. Subsequently, the surgical procedure for external jugular vein catheterization is conducted to allow for daily intravenous drug treatment or repeated blood sampling via a subcutaneous vascular access button. Despite the two dorsal implants, the monitoring of AAA development is readily facilitated by sequential semi-automated 3D ultrasound analysis, which yields comprehensive information on the expansion of aortic diameter and volume and on aneurysm morphology, as illustrated by experimental examples.

Objective: The purpose of this study was to assess the associations between malignancy, therapeutic regimens, and aorto-iliac aneurysm (i.e., abdominal aortic aneurysm [AAA]) growth rates., Methods: A retrospective single centre analysis identified patients with an AAA plus cancer. Patients who had two or more computed tomography angiograms over six months or more and additional malignancy were included. Clinical data and aneurysm diameters were analysed. AAA growth under cancer therapy (chemotherapy or radiation) was compared with a non-cancer AAA control cohort and to meta-analysis data. Statistics included t tests and a linear regression model with correction for initial aortic diameter and type of treatment., Results: From 2003 to 2020, 217 patients (median age 70 years; 92% male) with 246 aneurysms (58.8% AAA) and 238 malignancies were identified. Prostate (26.7%) and lung (15.7%) cancer were most frequently seen. One hundred and fifty-seven patients (72.3%) received chemotherapy, 105 patients (48.4%) radiation, and 79 (36.4%) both. Annual AAA growth (mean ± standard deviation) was not statistically significantly different for cancer and non-cancer patients (2.0 ± 2.3 vs. 2.8 ± 2.1 mm/year; p = .20). However, subgroup analyses revealed that radiation was associated with a statistically significantly reduced mean aneurysm growth rate compared with cancer patients without radiation (1.1 ± 1.3 vs. 1.6 ± 2.1 mm/year; p = .046) and to the non-cancer control cohort (1.7 ± 1.9 vs. 2.8 ± 2.1 mm/year; p = .007). Administration of antimetabolites resulted in statistically significantly increased AAA growth (+ 0.9 mm/year; p = .011), while topoisomerase inhibitors (- 0.8 mm/year; p = .17) and anti-androgens (- 0.5 mm/year; p = .27) showed a possible trend for reduced growth. Similar observations were noted for iliac aneurysms (n = 85). Additionally, the effects persisted for chemotherapy combinations (2.6 ± 1.4 substances/patient)., Conclusion: Patients with cancer and concomitant aortic aneurysms may require intensified monitoring when undergoing specific therapies, such as antimetabolite treatment, as they may experience an increased aneurysm growth rate. Radiation may be associated with reduced aneurysm growth., (Copyright © 2022 European Society for Vascular Surgery. Published by Elsevier B.V. All rights reserved.)

Background: Available mouse models for abdominal aortic aneurysms (AAAs) differ substantially in the applied triggers, associated pathomechanisms and rate of vessel expansion. While maximum aortic diameter (determined after aneurysm excision or by 2D ultrasound) is commonly applied to document aneurysm development, we evaluated the sensitivity and reproducibility of 3D ultrasound to monitor aneurysm growth in four distinct mouse models of AAA., Methods: The models included angiotensin-II infusion in ApoE deficient mice, topical elastase application on aortas in C57BL/6J mice (with or without oral administration of β-aminoproprionitrile) and intraluminal elastase perfusion in C57BL/6J mice. AAA development was monitored using semi-automated 3D ultrasound for aortic volume calculation over 12 mm length and assessment of maximum aortic diameter., Results: While the models differed substantially in the time course of aneurysm development, 3D ultrasound measurements (volume and diameter) proved highly reproducible with concordance correlation coefficients > 0.93 and variations below 9% between two independent observers. Except for the elastase perfusion model where aorta expansion was lowest and best detected by diameter increase, all other models showed high sensitivity of absolute volume and diameter measurements in monitoring AAA formation and progression by 3D ultrasound. When compared to standard 2D ultrasound, the 3D derived parameters generally reached the highest effect size., Conclusion: This study has yielded novel information on the robustness and limitations of semi-automated 3D ultrasound analysis and provided the first direct comparison of aortic volume increase over time in four widely applied mouse models of AAA. While 3D ultrasound generally proved highly sensitive in detecting early AAA formation, the 3D based volume analysis was found inferior to maximum diameter assessment in the elastase perfusion model where the extent of inflicted local injury is determined by individual anatomical features., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ibrahim, Bleichert, Klopf, Kurzreiter, Knöbl, Hayden, Busch, Stiglbauer-Tscholakoff, Eilenberg, Neumayer, Bailey and Brostjan.)

Objective: The maximal aortic diameter is currently the only clinically applied predictor of abdominal aortic aneurysm (AAA) progression. It is known that the risk of rupture is associated with aneurysm size; hence, accurate monitoring of AAA expansion is crucial. Aneurysmal vessel wall calcification and its implication on AAA expansion are insufficiently explored. We evaluated the vascular calcification using longitudinal computed tomography angiographies (CTA) of patients with an AAA and its association with AAA growth., Methods: We conducted a retrospective study of 102 patients with an AAA with a total of 389 abdominal CTAs at 6-month intervals, treated and followed at the Division of Vascular Surgery, Department of General Surgery, Medical University of Vienna. Digitally stored CTAs were reviewed for vascular calcification (volume and score) of the infrarenal aorta and common iliac arteries as well as for morphometric AAA analysis. In the prognostic setting, slow versus fast AAA progression was defined as a less than 2 mm or a 2-mm or greater increase in AAA diameter over 6 months. In addition, to analyze the association of vascular calcification and the AAA growth rate with longitudinal monitoring data, a specifically tailored log-linear mixed model was used., Results: An inverse relation of increased abdominal vessel wall calcification and short-term AAA progression was detected. Compared with fast progressing AAA, the median calcification volume of the infrarenal aorta (1225.3 mm³ vs 519.8 mm³; P = .003), the median total calcification volume (2014.1 mm³ vs 1434.9 mm³; P = .008), and the median abdominal total customized Agatston calcium (cAC) score (1663.5 vs 718.4; P = .003) were significantly increased in slow progressing AAA. Importantly, a log-linear mixed model efficiently predicted AAA expansion based on current diameter and abdominal total cAC score (P = .042)., Conclusions: We assessed the prognostic value of CTA-measured vascular calcification for AAA progression. Increased vascular calcification stabilizes the aortic aneurysmal wall and likely protects against progressive AAA expansion, resulting in a significant decrease of aneurysm growth over time. As a consequence, this may have implications for rupture risk, mortality, morbidity, and cost., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Sporadic apoptosis of tumour cells is a commonly observed feature of colorectal cancer (CRC) and strongly correlates with adverse patient prognosis. The uptake of apoptotic cell debris by neutrophils induces a non-inflammatory, pro-regenerative, and hence potentially pro-tumorigenic phenotype. In this study, we therefore sought to investigate the impact of apoptotic CRC cells on neutrophils and its consequence on other immune cells of the tumour microenvironment. Apoptosis induced by combined TNFα-treatment and UV-C irradiation, as well as various chemotherapeutic agents, led to a substantial release of neutrophil-attracting chemokines, most importantly interleukin-8 (IL-8), in both primary patient-derived and established CRC cells. Accordingly, conditioned media of apoptotic tumour cells selectively stimulated chemotaxis of neutrophils, but not T cells or monocytes. Notably, caspase-inhibition partially reduced IL-8 secretion, suggesting that caspase activity might be required for apoptosis-induced IL-8 release. Moreover, apoptotic tumour cell-conditioned media considerably prolonged neutrophil lifespan and induced an activated CD66b high CD11b high CD62L low phenotype, comparable to that of tumour-associated neutrophils in CRC patients, as assessed by flow cytometry of dissociated CRC tissues. Immunohistochemical analyses of 35 CRC patients further revealed a preferential accumulation of neutrophils at sites of apoptotic tumour cells defined by the expression of epithelial cell-specific caspase-cleaved cytokeratin-18. The same areas were also highly infiltrated by macrophages, while T cells were virtually absent. Notably, neutrophils induced an M2-like CD86 low CD163 + CD206 + phenotype in co-cultured monocyte-derived macrophages and suppressed LPS-induced pro-inflammatory cytokine release. In an in vitro transwell model, IL-8 blockade efficiently prevented neutrophil-induced anti-inflammatory macrophage polarisation by inhibiting neutrophil migration towards IL-8 gradients generated by apoptotic CRC cells. To conclude, our data suggest that apoptotic cancer cells release chemotactic factors that attract neutrophils into the tumour, where their interaction with neighbouring macrophages might promote an immunologically unfavourable tumour microenvironment. This effect may contribute to tumour recurrence after chemotherapy-induced apoptosis., (© 2022. The Author(s).)

Background: The most relevant determinant in scheduling monitoring intervals for abdominal aortic aneurysms (AAAs) is maximum diameter. The aim of the study was to develop a statistical model that takes into account specific characteristics of AAA growth distributions such as between-patient variability as well as within-patient variability across time, and allows probabilistic statements to be made regarding expected AAA growth., Methods: CT angiography (CTA) data from patients monitored at 6-month intervals with maximum AAA diameters at baseline between 30 and 66 mm were used to develop the model. By extending the model of geometric Brownian motion with a log-normal random effect, a stochastic growth model was developed. An additional set of ultrasound-based growth data was used for external validation., Results: The study data included 363 CTAs from 87 patients, and the external validation set comprised 390 patients. Internal and external cross-validation showed that the stochastic growth model allowed accurate description of the distribution of aneurysm growth. Median relative growth within 1 year was 4.1 (5-95 per cent quantile 0.5-13.3) per cent. Model calculations further resulted in relative 1-year growth of 7.0 (1.0-16.4) per cent for patients with previously observed rapid 1-year growth of 10 per cent, and 2.6 (0.3-8.3) per cent for those with previously observed slow growth of 1 per cent. The probability of exceeding a threshold of 55 mm was calculated to be 1.78 per cent at most when adhering to the current RESCAN guidelines for rescreening intervals. An online calculator based on the fitted model was made available., Conclusion: The stochastic growth model was found to provide a reliable tool for predicting AAA growth., (© The Author(s) 2021. Published by Oxford University Press on behalf of BJS Society Ltd.)

Background: Micro-metastatic growth is considered the main source of early cancer recurrence. Nutritional and microenvironmental components are increasingly recognized to play a significant role in the liver. We explored the predictive potential of preoperative plasma metabolites for postoperative disease recurrence in colorectal cancer liver metastasis (CRCLM) patients., Methods: All included patients (n = 71) had undergone R0 liver resection for colorectal cancer liver metastasis in the years between 2012 and 2018. Preoperative blood samples were collected and assessed for 180 metabolites using a preconfigured mass-spectrometry kit (Biocrates Absolute IDQ p180 kit). Postoperative disease-free (DFS) and overall survival (OS) were prospectively recorded. Patients that recurred within 6 months after surgery were defined as "high-risk" and, subsequently, a three-metabolite model was created which can assess DFS in our collective., Results: Multiple lysophosphatidylcholines (lysoPCs) and phosphatidylcholines (PCs) significantly predicted disease recurrence within 6 months (strongest: PC aa C36:1 AUC = 0.83, p = 0.003, PC ae C34:0 AUC = 0.83, p = 0.004 and lysoPC a C18:1 AUC = 0.8, p = 0.006). High-risk patients had a median DFS of 183 days versus 522 days in low-risk population (p = 0.016, HR = 1.98 95% CI 1.16-4.35) with a 6 months recurrence rate of 47.6% versus 4.7%, outperforming routine predictors of oncological outcome., Conclusion: Circulating metabolites identified CRCLM patients at highest risk for 6 months disease recurrence after surgery. Our data also suggests that circulating metabolites might play a significant pathophysiological role in micro-metastatic growth and concomitant early tumor recurrences after liver resection. However, the clinical applicability and performance of this proposed metabolomic concept needs to be independently validated in future studies., (Copyright © 2021 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

In this observational case-control study, circulating levels of complement factors C3a and C5a and leukotriene B4 (LTB4) were analysed in abdominal aortic aneurysm (AAA) patients regarding their association with diagnosis and prognosis. Serum C5a was significantly raised in AAA patients compared to healthy controls-median 84.5 ng/ml (IQR = 37.5 ng/ml) vs. 67.7 ng/ml (IQR = 26.2 ng/ml), p = 0.007-but was not elevated in patients with athero-occlusive disease. Serum C5a levels correlated significantly with the increase in maximum AAA diameter over the following 6 months (r = 0.319, p = 0.021). The median growth in the lowest quartile of C5a (< 70 ng/ml) was 50% less compared to the highest C5a quartile (> 101 ng/ml): 1.0 mm/6 months (IQR = 0.8 mm) vs. 2.0 mm/6 months (IQR = 1.5 mm), p = 0.014. A log-linear mixed model predicted AAA expansion based on current diameter and C5a level. To our knowledge, this is the first study linking complement activation, in particular C5a serum level, with AAA progression., (© 2020. The Author(s).)

Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of abdominal aortic aneurysms (AAAs). This study has addressed the notion that NET components might serve as AAA biomarkers or novel targets of AAA therapy. Thus, parameters of neutrophil activation and NET formation were measured in plasma. Their diagnostic marker value was explored in 41 AAA patients and 38 healthy controls. The NET parameter citrullinated histone H3 (citH3) was then validated in 63 AAA patients and 63 controls matched for cardiovascular disease. The prognostic marker potential was investigated in 54 observation periods of AAA growth over 6 months. NETs were further assessed in conditioned medium and sections of aortic tissue. CitH3 was found to be increased in blood (median 362 vs 304 ng/mL, P = 0.004) and aortic tissue (50 vs 1.5 ng/mg, P < 0.001) of AAA patients compared to healthy controls and accumulated in the intraluminal thrombus (629 ng/mg). The diagnostic potential of citH3 ranged at 0.705 area under the ROC curve (AUROC) and was validated with the independent sample set. Furthermore, plasma citH3 predicted AAA growth over the next 6 months (AUROC: 0.707, P = 0.015) and dropped significantly after surgical aneurysm repair. In an angiotensin II - based mouse model of experimental AAA, an inhibitor of histone citrullination was applied to block NET formation and AAA progression. Of note, further growth of an established aneurysm was prevented in mice treated with the NET inhibitor (P = 0.040). In conclusion, histone citrullination represents a promising AAA biomarker and potential therapeutic target to control disease progression., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Abdominal aortic aneurysm (AAA) is a condition that has considerable socioeconomic impact and an eventual rupture is associated with high mortality and morbidity. Despite decades of research, surgical repair remains the treatment of choice and no medical therapy is currently available. Animal models and, in particular, murine models, of AAA are a vital tool for experimental in vivo research. However, each of the different models has individual limitations and provide only partial mimicry of human disease. This narrative review addresses the translational potential of the available mouse models, highlighting unanswered questions from a clinical perspective. It is based on a thorough presentation of the available literature and more than a decade of personal experience, with most of the available models in experimental and translational AAA research., Results: From all the models published, only the four inducible models, namely the angiotensin II model (AngII), the porcine pancreatic elastase perfusion model (PPE), the external periadventitial elastase application (ePPE), and the CaCl 2 model have been widely used by different independent research groups. Although the angiotensin II model provides features of dissection and aneurysm formation, the PPE model shows reliable features of human AAA, especially beyond day 7 after induction, but remains technically challenging. The translational value of ePPE as a model and the combination with β-aminopropionitrile to induce rupture and intraluminal thrombus formation is promising, but warrants further mechanistic insights. Finally, the external CaCl 2 application is known to produce inflammatory vascular wall thickening. Unmet translational research questions include the origin of AAA development, monitoring aneurysm growth, gender issues, and novel surgical therapies as well as novel nonsurgical therapies., Conclusion: New imaging techniques, experimental therapeutic alternatives, and endovascular treatment options provide a plethora of research topics to strengthen the individual features of currently available mouse models, creating the possibility of shedding new light on translational research questions., (© 2021 by the Society for Vascular Surgery. Published by Elsevier Inc.)

Here, we describe a novel approach that allows pathologists to three-dimensionally analyse malignant tissues, including the tumour-host tissue interface. Our visualization technique utilizes a combination of ultrafast chemical tissue clearing and light-sheet microscopy to obtain virtual slices and 3D reconstructions of up to multiple centimetre sized tumour resectates. For the clearing of tumours we propose a preparation technique comprising three steps: (a) Fixation and enhancement of tissue autofluorescence with formalin/5-sulfosalicylic acid. (b) Ultrafast active chemical dehydration with 2,2-dimethoxypropane and (c) refractive index matching with dibenzyl ether at up to 56 °C. After clearing, the tumour resectates are imaged. The images are computationally post-processed for contrast enhancement and artefact removal and then 3D reconstructed. Importantly, the sequence a-c is fully reversible, allowing the morphological correlation of one and the same histological structures, once visualized with our novel technique and once visualized by standard H&E- and IHC-staining. After reverting the clearing procedure followed by standard H&E processing, the hallmarks of ductal carcinoma in situ (DCIS) found in the cleared samples could be successfully correlated with the corresponding structures present in H&E and IHC staining. Since the imaging of several thousands of optical sections is a fast process, it is possible to analyse a larger part of the tumour than by mechanical slicing. As this also adds further information about the 3D structure of malignancies, we expect that our technology will become a valuable addition for histological diagnosis in clinical pathology.

Objective: Macrophages are immune cells, capable to remodel the extracellular matrix, which can harbor extracellular DNA incorporated into neutrophil extracellular traps (NETs). To study the breakdown of NETs we studied the capability of macrophage subsets to degrade these structures in vitro and in vivo in a murine thrombosis model. Furthermore, we analyzed human abdominal aortic aneurysm samples in support of our in vitro and in vivo results. Approach and Results: Macrophages were seeded onto blood clots or isolated NETs and polarized. All macrophages were capable to degrade NETs. For initial breakdown, macrophages relied on extracellular deoxyribonucleases. Proinflammatory polarization enhanced NET degradation. The boost in degradation was because of increased macropinocytosis, as inhibition by imipramine diminished their NET breakdown. Inhibition of macropinocytosis in a murine thrombosis model led to increased NET burden and reduced thrombus resolution in vivo. When analyzing abdominal aortic aneurysm samples, macrophage density furthermore corresponded negatively with the amount of local NETs in the intraluminal thrombi as well as in the vessel wall, as increased macrophage density was associated with a reduction in NET burden., Conclusions: We provide evidence that macrophages degrade NETs by extracellular predigestion and subsequent uptake. Furthermore, we show that proinflammatory macrophages increase NET degradation through enhanced macropinocytosis, priming them for NET engulfment. Based on our findings, that inhibition of macropinocytosis in mice corresponded to increased NET amounts in thrombi and that local macrophage density in human abdominal aortic aneurysm is negatively associated with surrounding NETs, we hypothesize, that macrophages are able to degrade NETs in vivo.

Von Willebrand factor (VWF) plays a major role in arterial thrombosis. Antiplatelet drugs induce only a moderate relative risk reduction after atherothrombosis, and their inhibitory effects are compromised under high shear rates when VWF levels are increased. Therefore, we investigated the ex vivo effects of a third-generation anti-VWF aptamer (BT200) before/after stimulated VWF release. We studied the concentration-effect curves BT200 had on VWF activity, platelet plug formation under high shear rates (PFA), and ristocetin-induced platelet aggregation (Multiplate) before and after desmopressin or endotoxin infusions in healthy volunteers. VWF levels increased > 2.5-fold after desmopressin or endotoxin infusion (p < 0.001) and both agents elevated circulating VWF activity. At baseline, 0.51 µg/ml BT200 reduced VWF activity to 20% of normal, but 2.5-fold higher BT200 levels were required after desmopressin administration (p < 0.001). Similarly, twofold higher BT200 concentrations were needed after endotoxin infusion compared to baseline (p < 0.011). BT200 levels of 0.49 µg/ml prolonged collagen-ADP closure times to > 300 s at baseline, whereas 1.35 µg/ml BT200 were needed 2 h after desmopressin infusion. Similarly, twofold higher BT200 concentrations were necessary to inhibit ristocetin induced aggregation after desmopressin infusion compared to baseline (p < 0.001). Both stimuli elevated plasma VWF levels in a manner representative of thrombotic or pro-inflammatory conditions such as arterial thrombosis. Even under these conditions, BT200 potently inhibited VWF activity and VWF-dependent platelet function, but higher BT200 concentrations were required for comparable effects relative to the unstimulated state.

TLRs are a family of signaling sensors that play a crucial role in the host immune response and are involved in the modulation of inflammatory processes. To study their contribution to abdominal aortic aneurysm (AAA) formation and development, we determined the frequency of TLR2 , TLR3 , TLR4 , and TLR9 single-nucleotide polymorphisms (SNPs) and investigated the association between polymorphisms and the risk of AAA incidence. A total of 104 patients with AAAs and 112 healthy, unrelated volunteers were screened for the presence of TLR2 (2029C/T and 2258G/A), TLR3 (1377C/T, 1234C/T, and -7C/A), TLR4 (896A/G, 1196C/T, and 3266G/A), and TLR9 (-1237T/C, -1486T/C, 1174G/A, and 2848C/T) SNPs by using PCR-RFLP analysis. The heterozygous genotype of the TLR2 2029C/T SNP was more common in patients with AAA than in healthy subjects ( p < 0.0001) and was associated with at least an 8-fold increased risk of AAA incidence ( p < 0.001). The wild-type genotype of the TLR3 -7C/A SNP was associated with a 3-fold increased risk of hypertension ( p = 0.026). The heterozygous TLR3 genotype 1377C/T and -7C/A SNPs were less common in patients with AAA than in healthy subjects ( p < 0.0001 and p = 0.0004, respectively) and were associated with a decreased risk of AAA occurrence ( p < 0.001 and p = 0.0012, respectively). No relation to AAA risk was found for TLR4 SNPs. Heterozygous genotypes of the TLR2 2029C/T and TLR3 1377C/T and -7C/A SNPs may serve as genetic biomarkers of AAA incidence., (Copyright © 2020 by The American Association of Immunologists, Inc.)

The lifespan of a neutrophil is short and limited by programmed cell death, followed by efferocytosis. When activated or exposed to insult, neutrophil death may be delayed to support neutrophil effector functions such as phagocytosis, cytokine release, and pathogen destruction by degranulation. However, neutrophils may also alter the type of cell death and thereby affect inflammatory responses and tissue remodeling. This review briefly introduces the various forms of neutrophil death including apoptosis, necrosis/necroptosis, and the formation of so-called "neutrophil extracellular traps" (NETs), and it summarizes the clearance of dead cells by efferocytosis. Importantly, distinct types of neutrophil death have been found to drive chronic inflammatory disorders and cancer. Thus, the tumor and its microenvironment can delay neutrophil apoptosis to exploit their pro-angiogenic and pro-metastatic properties. Conversely, neutrophils may enter rapid and suicidal cell death by forming extracellular traps, which are expelled DNA strands with neutrophil proteins. Components of these DNA-protein complexes such as histones, high-mobility group protein B1, or neutrophil elastase have been found to promote cancer cell proliferation, adhesion, migration, invasion, and thereby tumor metastasis. In other settings of chronic inflammatory disease such as gout, NETs have been found protective rather than detrimental, as they promoted the local degradation of pro-inflammatory cytokines by neutrophil proteases. Thus, the interaction of neutrophils with the tissue environment extends beyond the stage of the living cell and the type of neutrophil death shapes immune responses and tissue remodeling in health and disease., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2020.)

Recently, von-Willebrand-Factor (vWF) has been shown to correlate with postoperative liver dysfunction (LD). Accordingly, "disintegrin-like metalloprotease with thrombospondin type1 motif" (ADAMTS13) is known to cleave vWF in less active fragments. Thus, we aimed to evaluate the diagnostic potential of ADAMTS13-activity (ADAMTS13-AC) to identify patients with postoperative LD after hepatectomy. Accordingly 37 patients undergoing hepatectomy for different neoplastic entities were included in this study. Plasma ADAMTS13-AC and vWF-Ag were measured 1 day prior to (preOP), 1 and 5 days (POD1/5) after hepatectomy. In accordance to the ISGLS-criteria LD was prospectively recorded. In this context, perioperative ADAMTS13-AC- and vWF-Ag/ADAMTS13-AC-ratio- levels revealed a significant increase after hepatectomy. Accordingly, elevated vWF-Ag/ADAMTS13-AC-ratio significantly predicted LD (preOP AUC: 0.75, p = 0.02; POD1 AUC: 0.80, p = 0.03). Patients who fulfilled our perioperative vWF-Ag/ADAMTS13-AC-ratio cut-off-levels (preOP: ≥116, POD1: ≥165) suffered from significantly higher incidences of LD (preOP: 70% vs. 30%, p = 0.01; POD1: 83% vs. 17%, p = 0.001). In conclusion, perioperative ADAMTS13-AC measurement may serve as a useful parameter to early detect high-risk patients developing postoperative LD prior to liver resection in patients suffering from hepatic malignancies. Indeed, further investigations have to be performed to consolidate its role as a predictive marker for LD.

The p21-activated kinases (PAKs) are key nodes in oncogenic signalling pathways controlling growth, survival, and motility of cancer cells. Their activity is increased in many human cancers and is associated with poor prognosis. To date, PAK deregulation has mainly been studied in solid tumours, where PAK1 and PAK4 are the main isoforms deregulated. We show that PAK1 and PAK2 are the critical isoforms in a BCR/ABL1 + haematopoietic malignancy. In suspension, leukaemic cells deficient for PAK1 and PAK2 undergo apoptosis, while the loss of either protein is well tolerated. Transfer of medium conditioned by shPAK2- but not shPAK1-expressing leukaemic cells interferes with endothelial cell growth. We found that leukaemic cells produce exosomes containing PAK2. Transfer of isolated exosomes supports endothelial cell proliferation. In parallel, we found that leukaemic cells explicitly require PAK2 to grow towards an extracellular matrix. PAK2-deficient cells fail to form colonies in methylcellulose and to induce lymphomas in vivo. PAK2 might therefore be the critical isoform in leukaemic cells by controlling tumour growth in a dual manner: vascularization via exosome-mediated transfer to endothelial cells and remodelling of the extracellular matrix. This finding suggests that the PAK2 isoform represents a promising target for the treatment of haematological diseases., (© 2017 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.)

Granulocyte colony-stimulating factor (G-CSF) is an activator of neutrophil granulocytes. Neutrophil extracellular traps are a defensive mechanism consisting of neutrophils, platelets, DNA, histones and antimicrobial proteins. This study was performed to determine whether G-CSF increases histone-complexed DNA in the plasma of healthy volunteers. In total, 51 healthy volunteers (25 males and 26 females) were treated with G-CSF (18 with 300 µg single dose i.v., 27 with 5 µg/kg s.c. for 4 days) and six participants received a placebo. Histone-complexed DNA was measured by enzyme immunoassay in plasma samples at predefined time points (0, 2, 4, 6, 24 h after single dose, day 1, day 2 and day 5 after repeated doses). Histone levels were quantified by Western blotting. A single dose of G-CSF rapidly increased hc-DNA by about 50 % (p < 0.05 for 2-24 h). After repeated doses the increase was even more pronounced: hc-DNA increased by about 50 % (3.0 ± 0.9, p < 0.001 after 24 h and about fourfold after 96 h (p < 0.001)). A statistical significant increase in histone levels was detected as early as 4 h after G-CSF injection (0.43 ± 0.2 vs. 1.08 ± 0.3 µg/ml; p = 0.034). In the placebo group no significant changes occurred. Moreover, significantly higher levels of hc-DNA were measured in male compared to female subjects (226 ± 43 vs. 84 ± 19, p < 0.001). G-CSF injection substantially increases hc-DNA levels in healthy volunteers. There is a significant gender difference in hc-DNA at the baseline.

Venous thromboembolism (VTE) is common in patients with brain tumors, and underlying mechanisms are unclear. We hypothesized that podoplanin, a sialomucin-like glycoprotein, increases the risk of VTE in primary brain tumors via its ability to induce platelet aggregation. Immunohistochemical staining against podoplanin and intratumoral platelet aggregates was performed in brain tumor specimens of 213 patients (mostly high-grade gliomas [89%]) included in the Vienna Cancer and Thrombosis Study, a prospective observational cohort study of patients with newly diagnosed cancer or progressive disease aimed at identifying patients at risk of VTE. Platelet aggregation in response to primary human glioblastoma cells was investigated in vitro. During 2-year follow-up, 29 (13.6%) patients developed VTE. One-hundred fifty-one tumor specimens stained positive for podoplanin (33 high expression, 47 medium expression, 71 low expression). Patients with podoplanin-positive tumors had lower peripheral blood platelet counts ( P < .001) and higher D-dimer levels ( P < .001). Podoplanin staining intensity was associated with increasing levels of intravascular platelet aggregates in tumor specimens ( P < .001). High podoplanin expression was associated with an increased risk of VTE (hazard ratio for high vs no podoplanin expression: 5.71; 95% confidence interval, 1.52-21.26; P = 010), independent of age, sex, and tumor type. Podoplanin-positive primary glioblastoma cells induced aggregation of human platelets in vitro, which could be abrogated by an antipodoplanin antibody. In conclusion, high podoplanin expression in primary brain tumors induces platelet aggregation, correlates with hypercoagulability, and is associated with increased risk of VTE. Our data indicate novel insights into the pathogenesis of VTE in primary brain tumors., (© 2017 by The American Society of Hematology.)

The oncogenic potential of the transcriptional repressor Bcl-6 (B-cell lymphoma 6) was originally discovered in non-Hodgkin patients and the soluble Bcl-6 inhibitor 79-6 was developed to treat diffuse large B-cell lymphomas with aberrant Bcl-6 expression. Since we found Bcl-6 and its co-repressor BCoR (Bcl-6 interacting co-repressor) to be regulated in human microvascular endothelium by colorectal cancer cells, we investigated their function in sprouting angiogenesis which is central to tumor growth. Based on Bcl-6/BCoR gene silencing we found that the transcriptional repressor complex in fact constitutes an endogenous inhibitor of vascular sprouting by supporting the stalk cell phenotype: control of Notch target genes (HES1, HEY1, DLL4) and cell cycle regulators (cyclin A and B1). Thus, when endothelial cells were transiently transfected with Bcl-6 and/or BCoR siRNA, vascular sprouting was prominently induced. Comparably, when the soluble Bcl-6 inhibitor 79-6 was applied in the mouse retina model of physiological angiogenesis, endothelial sprouting and branching were significantly enhanced. To address the question whether clinical treatment with 79-6 might therefore have detrimental therapeutic effects by promoting tumor angiogenesis, mouse xenograft models of colorectal cancer and diffuse large B-cell lymphoma were tested. Despite a tendency to increased tumor vessel density, 79-6 therapy did not enhance tumor expansion. In contrast, growth of colorectal carcinomas was significantly reduced which is likely due to a combined 79-6 effect on cancer cells and tumor stroma. These findings may provide valuable information regarding the future clinical development of Bcl-6 inhibitors.

Background: We report the results of a phase II trial of adding the anti-ascular endothelial growth factor (VEGF) bevacizumab to gemcitabine neoadjuvant chemotherapy for patients with borderline and unresectable non-metastatic pancreatic cancer., Patients and Methods: Patients were assigned to one of the two treatment arms. Both groups received 1,000 mg/m(2) gemcitabine on days 1, 8, and 15 of a 4-week cycle for a total of four cycles. Group 1 received 5 mg/kg bevacizumab for six weeks (three doses), every second week, starting at week 6 of gemcitabine therapy. Group 2 received 5 mg/kg bevacizumab for 12 weeks (six doses), every second week, starting at week 1 of gemcitabine therapy. The objective of the present study was to assess the rate of complete radical resection and overall survival., Results: A total of 30 patients were enrolled: 19 patients had unresectable and 11 patients had borderline-resectable pancreatic cancer. Eleven patients (37%) underwent resection. The median overall survival of patients who underwent tumor resection was 13 months (95% confidence interval=11-15 months)., Conclusion: In general, adding bevacizumab to neoadjuvant gemcitabine does not improve outcomes for patients with locally advanced pancreatic cancer. However, in individual cases, surgery is consequently possible and prolonged survival may be observed.

Immunotherapy with autologous dendritic cells (DCs) loaded with tumor lysate(s) from allogeneic tumor cell lines is a novel strategy to induce immune responses in cancer patients. We report on a pilot trial of autologous DCs pulsed with tumor cell lysate derived from allogeneic medullary thyroid carcinoma (MTC) cell lines in patients with metastatic MTC. The purpose of this study was to assess the safety, resulting immune responses and clinical activity of the DCs. DCs were injected into a groin lymph node at 3-week intervals. Monitoring included serial calcitonin tumor marker measurements, radiological imaging and immunological in vitro tests (T-cell interferon-gamma detection assay, T-cell cytotoxicity assay). Ten patients (median age 47 years, range 29-77) were enrolled. DC vaccinations were well-tolerated and safe. After a median follow-up of 11 months, (range 7-26), 3 (30%) of 10 patients had stable disease, while 7 (70%) of the patients progressed during treatment. In 2 patients with stable disease, calcitonin decreased below treatment levels, paralleled by a T-cell-mediated immune response. Notably, treatment with DCs pulsed with a combination of different tumor cell lysates was followed by a calcitonin decrease in 4 patients who had previously experienced a calcitonin increase during monotherapy with DCs pulsed with a single lysate. Allogeneic tumor cell lysate-based DC immunotherapy is well-tolerated and safe. Combined treatment with different tumor cell lysate-pulsed DCs increases the likelihood of a calcitonin tumor marker response and should therefore be preferred over monotherapy with DCs pulsed with a single lysate.

The objective of this study was to assess the toxicity and immunological response induced by autologous dendritic cells (DCs) pulsed with allogeneic tumor lysate in a pancreatic cancer patient. The lack of available tumor peptides in pancreatic cancer strongly supports the idea to use allogeneic tumor cells as a source of antigens. The patient suffering from a stage IV pancreatic cancer received 1-2x10(7) autologous monocyte-derived DCs in three-week intervals injected into a groin lymph node. Monocytes from peripheral blood were isolated by magnetic bead selection. For the first ten vaccinations DCs were loaded with autologous tumor cell lysate obtained during surgical exploration. After consumption of the autologous lysate, equal numbers of DCs were pulsed with lysate of the tumor cell line AsPc-1 and BxPc-3 for a further five vaccinations. Peripheral mononuclear cells (PMNCs) were harvested after the seventh and compared with PMNCs obtained after the fourteenth vaccination for immunological response. Delayed type hypersensitivity reactivity to DCs pulsed with autologous and allogeneic tumor lysate was also assessed. The patient received a total of fifteen vaccinations. There was no toxicity or evidence of autoimmunity observed. Delayed type hypersensitivity was found to be positive for the autologous as well as the allogeneic tumor lysate pulsed DCs. in vitro cytotoxicity assays demonstrated a dramatic increase of the PMNC killing capacity against the pancreatic cancer cell lines AsPc-1 and BxPc-3 after the fourteenth compared to the seventh vaccination. CT scans revealed a stable disease for six months. The administration of autologous DCs pulsed with allogeneic tumor lysate is non-toxic and suitable for inducing an immunological anti-tumor response. Even though this study was confined to a single patient, the data might open a door for novel immunotherapeutical strategies.