Dominic Schauer

Evidence for serotonin as a relevant inducer of liver regeneration after liver resection in humans

Liver regeneration (LR) involves a complex interplay of growth factors and antagonists. In this context, platelet‐derived serotonin (5‐HT) has been identified as a critical inducer of LR in mice. Clinical evidence for a role of 5‐HT in LR in humans is lacking. Accordingly, serum and plasma 5‐HT was monitored perioperatively in 60 patients undergoing liver resection, of which 35 served as exploration and 25 as validation sets. Intraplatelet (IP) levels of 5‐HT were calculated by subtraction of plasma 5‐HT from serum values. Serum markers of liver function were used to evaluate LR and liver dysfunction (LD). In the exploration setting, IP 5‐HT levels significantly decreased after liver resection (P < 0.001) and gradually recovered during the first week. IP 5‐HT measured before surgery specifically predicted LD in the subsequent 7 days (area under the curve: 0.721; P = 0.029). Patients suffering from postoperative LD and morbidity were found to have reduced IP 5‐HT levels during the entire perioperative period. Furthermore, we validated that reduced preoperative IP 5‐HT (<73 ng/mL) was associated with an increased incidence of postoperative LD and morbidity (P =0.045 and P = 0.021) and were able to demonstrate that IP 5‐HT levels were an independent predictor of poor clinical outcome. Conclusions: These findings provide evidence that IP 5‐HT correlates with LR in humans: Patients with low IP 5‐HT before liver resection suffered from delayed hepatic regeneration. Therefore, IP 5‐HT levels may prove a helpful clinical marker to predict postoperative LD and clinical outcome before hepatic resection and initiate suitable interventions. (Hepatology 2014;60:257‐266)

Intermediate Monocytes but Not TIE2-Expressing Monocytes Are a Sensitive Diagnostic Indicator for Colorectal Cancer

We have conducted the first study to determine the diagnostic potential of the CD14++CD16+ intermediate monocytes as compared to the pro-angiogenic subset of CD14++CD16+TIE2+ TIE2-expressing monocytes (TEMs) in cancer. These monocyte populations were investigated by flow cytometry in healthy volunteers (N = 32) and in colorectal carcinoma patients with localized (N = 24) or metastatic (N = 37) disease. We further determined blood levels of cytokines associated with monocyte regulation. The results revealed the intermediate monocyte subset to be significantly elevated in colorectal cancer patients and to show the highest frequencies in localized disease. Multivariate regression analysis identified intermediate monocytes as a significant independent variable in cancer prediction. With a cut-off value at 0.37% (intermediate monocytes of total leukocytes) the diagnostic sensitivity and specificity ranged at 69% and 81%, respectively. In contrast, TEM levels were elevated in localized cancer but did not differ significantly between groups and none of the cytokines correlated with monocyte subpopulations. Of interest, in vitro analyses supported the observation that intermediate monocytes were more potently induced by primary as opposed to metastatic cancer cells which may relate to the immunosuppressive milieu established in the advanced stage of metastatic disease. In conclusion, intermediate monocytes as compared to TIE2-expressing monocytes are a more sensitive diagnostic indicator of colorectal cancer.

Platelet-stored angiogenesis factors: Clinical monitoring is prone to artifacts

Background: The analysis of angiogenesis factors in the blood of tumor patients has given diverse results on their prognostic or predictive value. Since mediators of angiogenesis are stored in platelets, their measurement in plasma is sensitive to inadvertent platelet activation during blood processing. Methods: Variants of blood withdrawal and plasma preparation were evaluated by ELISA for the detection of TSP-1, PF-4, VEGF and PD-ECGF. A total of 22 pancreatic cancer patients and 29 healthy volunteers were evaluated. Results: Plasma preparation with the anticoagulant mix of citrate, theophylline, adenosine, dipyridamole (CTAD) and immediate blood processing at 4°C was required for reproducible measurements of TSP-1, PF-4 and VEGF. Blood collection by venflon or inadvertent hemolysis during blood withdrawal caused significantly elevated TSP-1 and PF4 values. When optimized plasma preparation was applied, a significant increase of TSP-1 and VEGF in cancer patients was detected (P = 0.006; P < 0.001). Conclusion: The reliable plasma analysis of circulating platelet-stored angiogenesis factors requires preparation with CTAD at 4°C and blood collection by butterfly needle. Suboptimal procedures of plasma preparation are commonly applied in clinical monitoring of angiogenesis parameters which may account for the differences in reported plasma values and may have masked their predictive or prognostic marker potential.

Plasma thrombospondin 1 as a predictor of postoperative liver dysfunction.

Liver regeneration following liver resection involves a complex interplay of growth factors and their antagonists. Thrombospondin 1 has recently been identified as a critical inhibitor of liver regeneration by the activation of transforming growth factor β1 in mice, and preliminary data seem to confirm its relevance in humans. This study aimed to confirm these observations in an independent validation cohort.

Clinical evidence for thrombospondin-1 as a relevant suppressor of liver regeneration

To the Editor: The most significant factor determining morbidity and mortality following hepatectomy is the ability of the remnant liver to regenerate. In this context, Hayashi et al. [1] recently reported on a previously unknown role of thrombospondin-1 (TSP-1) in liver regeneration (LR). During tissue remodeling, TSP-1 is released into the pericellular space to regulate cell survival and extracellular matrix remodeling [2]. A major TSP-1 function is the conversion of latent TGF-b1 (transforming growth factor-beta 1) to its active form [3]. Based on a mouse model of partial hepatectomy, Hayashi et al. [1] demonstrated that TSP-1 was rapidly released in response to liver resection and triggered TGF-b1 activation and signal transduction in hepatocytes as a mechanism to inhibit the proliferative hepatic response [4–5]. Accordingly, knockdown of TSP-1 expression resulted in a significant reduction of TGF-b1 signaling, which promoted the proliferation of hepatocytes and accelerated LR [1]. Thus, TSP-1 was proposed as an inhibitory element and a potential therapeutic target of LR. We are now able to present the first clinical data, which support the relevance of TSP-1 in LR based on a cohort of metastasized colorectal cancer (mCRC) patients undergoing hepatic resection of liver metastases. A total of 66 mCRC patients without preexisting liver disease were included. Blood was retrieved before surgery (pre OP) and on day 1 (POD1) or days 5–8 (POD5) after liver resection, and plasma was prepared with an optimized method [6–7]. Samples were assayed by ELISA for human TSP-1 (R&D Systems, Minneapolis, MN, USA) and TGF-b1 (eBioscience, San Diego, CA, USA). For statistical analysis, non-parametric tests (Mann–Whitney U test, Wilcoxon test and Spearman correlation) and the Fisher exact test were applied. A significant increase in TSP-1 (Fig. 1A) and TGF-b1 was observed on POD1 (p = 0.007 and p = 0.006, respectively), which dropped moderately until POD5 (p = 0.016 and p = 0.397). Circulating TSP-1 and TGF-b1 showed a highly significant correlation (k = 0.736, p <0.001). To further investigate whether a high postoperative release of TSP-1 or TGF-b1 was associated with delayed LR, we assessed the incidence of postoperative liver dysfunction (LD), based on the previously defined ‘‘50–50 criteria’’ [8], with a prothrombin time (PT) <50% and a serum bilirubin (SB) level >50 lmol/L (>2.9 mg/dl). Our classification referred to measurements of any day within the first postoperative week to identify patients with delayed postoperative hepatic recovery. TSP-1 concentrations were significantly elevated (Fig. 1B) in patients with LD (median TSP-1 for LD: 98.8 ng/ml; no LD: 44.6 ng/ml, p = 0.003). Comparably, circulating TGF-b1 tended to be increased in individuals with LD (median TGF-b1 for LD: 2266 pg/ml; no LD: 1726 pg/ml), but the difference was not statistically significant (p = 0.102). Of note, our analyses were restricted to total TGF-b1 in patient blood since activated TGF-b1 was below ELISA detection limit. This may explain the lack of association between TGF-b1 levels and postoperative LD. To further characterize the potential of TSP-1 to predict postoperative LD, a ROC analysis was performed (Fig. 1C), revealing a significant predictive value of circulating TSP-1 on POD1 (AUC: 0.870; p = 0.003). A cut-off level of 100 ng/ml TSP-1 on POD1 was deduced to determine the high-risk group for postoperative LD with 95% specificity. No statistically significant difference in age and sex distribution or the site of primary tumor, type of hepatic resection and preoperative liver function parameters was observed between the TSP-1 individuals (>100 ng/ml TSP-1) and patients with lower circulating TSP-1 levels on POD1. When biochemical markers of postoperative liver function were compared between these groups, the TSP-1 patients showed a significant elevation in SB (Fig. 1D) and c-glutamyltransferase (p <0.001; p = 0.011). Prothrombin time (Fig. 1E) was found to be significantly decreased in TSP-1 patients (p <0.001). A trend towards elevated alanine and aspartate

Neoadjuvant bevacizumab persistently inactivates VEGF at the time of surgery despite preoperative cessation

Background:When anti-VEGF (vascular endothelial growth factor) antibody bevacizumab is applied in neoadjuvant treatment of colorectal cancer patients with liver metastasis, 5–6 weeks between last bevacizumab dose and liver resection are currently recommended to avoid complications in wound and liver regeneration. In this context, we aimed to determine whether VEGF is inactivated by bevacizumab at the time of surgery.Methods:Fifty colorectal cancer patients with liver metastases received neoadjuvant chemotherapy±bevacizumab supplementation. The last dose of bevacizumab was administered 6 weeks before surgery. Plasma, subcutaneous and intraabdominal wound fluid were analysed for VEGF content before and after liver resection (day 1–3). Immunoprecipitation was applied to determine the amount of bevacizumab-bound VEGF.Results:Bevacizumab-treated individuals showed no increase in perioperative complications. During the entire monitoring period, plasma VEGF was inactivated by bevacizumab. In wound fluid, VEGF was also completely bound by bevacizumab and was remarkably low compared with the control chemotherapy group.Conclusion:These data document that following a cessation time of 6 weeks, bevacizumab is fully active and blocks circulating and local VEGF at the time of liver resection. However, despite effective VEGF inactivation no increase in perioperative morbidity is recorded suggesting that VEGF activity is not essential in the immediate postoperative recovery period.

Monocytes with angiogenic potential are selectively induced by liver resection and accumulate near the site of liver regeneration

BackgroundMonocytes reportedly contribute to liver regeneration. Three subsets have been identified to date: classical, intermediate, non-classical monocytes. The intermediate population and a subtype expressing TIE2 (TEMs) were suggested to promote angiogenesis. In a clinical setting, we investigated which monocyte subsets are regulated after liver resection and correlate with postoperative liver function.MethodsIn 38 patients monocyte subsets were evaluated in blood and subhepatic wound fluid by flow cytometry before and 1-3 days after resection of colorectal liver metastases. The monocyte-regulating cytokines macrophage colony stimulating factor (M-CSF), transforming growth factor beta 1 (TGFβ1), and angiopoietin 2 (ANG-2) were measured in patient plasma by ELISA. C-reactive protein (CRP) and liver function parameters were retrieved from routine hospital analyses.ResultsOn post-operative day (POD) 1 blood monocytes shifted to significantly elevated levels of intermediate monocytes. In wound fluid, a delayed surge in intermediate monocytes was detected by POD 3. Furthermore, TEMs were highly enriched in wound fluid as compared to circulation. CRP and M-CSF levels were substantially increased in patient blood after surgery and correlated significantly with the frequency of intermediate monocytes. In addition, liver function parameters showed a significant association with intermediate monocyte levels on POD 3.ConclusionsThe reportedly pro-angiogenic subsets of monocytes are selectively increased upon liver resection and accumulate next to the site of liver regeneration. As previously proposed by in vitro experiments, the release of CRP and M-CSF may trigger the induction of intermediate monocytes. The correlation with liver parameters points to a functional involvement of these monocyte populations in liver regeneration which warrants further investigation.

The VEGF rise in blood of bevacizumab patients is not based on tumor escape but a host-blockade of VEGF clearance

Vascular endothelial growth factor (VEGF) has become a major target in cancer treatment as it promotes tumor angiogenesis. Therapy with anti-VEGF antibody bevacizumab reportedly induces high levels of circulating VEGF which may potentially contribute to resistance. Based on animal or computational models, mechanisms of VEGF induction by bevacizumab have been proposed but not verified in the clinical setting. Hence, we evaluated sixty patients with colorectal cancer metastases for changes in plasma VEGF during neoadjuvant/conversion and adjuvant chemotherapy with or without bevacizumab. VEGF expression was assessed in tissue sections of liver metastases. The VEGF source was investigated with in vitro cultures of tumor, endothelial cells, fibroblasts and platelets, and potential protein stabilization due to anti-VEGF therapy was addressed. A VEGF rise was observed in blood of bevacizumab patients but not in chemotherapy controls, and VEGF was found to be largely complexed by the antibody. A comparable VEGF increase occurred in the presence (neoadjuvant) and absence of the tumor (adjuvant). Accordingly, VEGF expression in tumor tissue was not determined by bevacizumab treatment. Investigations with isolated cell types did not reveal VEGF production in response to bevacizumab. However, antibody addition to endothelial cultures led to a dose-dependent blockade of VEGF internalization and hence stabilized VEGF in the supernatant. In conclusion, the VEGF rise in cancer patients treated with bevacizumab is not originating from the tumor. The accumulation of primarily host-derived VEGF in circulation can be explained by antibody interference with receptor-mediated endocytosis and protein degradation. Thus, the VEGF increase in response to bevacizumab therapy should not be regarded as a tumor escape mechanism.

Overexpression of the Transcriptional Repressor Complex BCL-6/BCoR Leads to Nuclear Aggregates Distinct from Classical Aggresomes

Nuclear inclusions of aggregated proteins have primarily been characterized for molecules with aberrant poly-glutamine repeats and for mutated or structurally altered proteins. They were termed “nuclear aggresomes” and misfolding was shown to promote association with molecular chaperones and proteasomes. Here, we report that two components of a transcriptional repressor complex (BCL-6 and BCoR) of wildtype amino acid sequence can independently or jointly induce the formation of nuclear aggregates when overexpressed. The observation that the majority of cells rapidly downregulate BCL-6/BCoR levels, supports the notion that expression of these proteins is under tight control. The inclusions occur when BCL-6/BCoR expression exceeds 150-fold of endogenous levels. They preferentially develop in the nucleus by a gradual increase in aggregate size to form large, spheroid structures which are not associated with heat shock proteins or marked by ubiquitin. In contrast, we find the close association of BCL-6/BCoR inclusions with PML bodies and a reduction in aggregation upon the concomitant overexpression of histone deacetylases or heat shock protein 70. In summary, our data offer a perspective on nuclear aggregates distinct from classical “nuclear aggresomes”: Large complexes of spheroid structure can evolve in the nucleus without being marked by the cellular machinery for protein refolding and degradation. However, nuclear proteostasis can be restored by balancing the levels of chaperones.

Chemotherapy of colorectal liver metastases induces a rapid rise in intermediate blood monocytes which predicts treatment response

ABSTRACT We have previously reported that intermediate monocytes (CD14++/CD16+) were increased in colorectal cancer (CRC) patients, while the subset of pro-angiogenic TIE2-expressing monocytes (TEMs) was not significantly elevated. This study was designed to evaluate changes in frequency and function of intermediate monocytes and TEMs during chemotherapy and anti-angiogenic cancer treatment and their relation to treatment response. Monocyte populations were determined by flow cytometry in 60 metastasized CRC (mCRC) patients who received neoadjuvant chemotherapy with or without bevacizumab. Blood samples were taken before treatment, after two therapy cycles, at the end of neoadjuvant therapy and immediately before surgical resection of liver metastases. Neoadjuvant treatment resulted in a significant increase in circulating intermediate monocytes which was most pronounced after two cycles and positively predicted tumor response (AUC = 0.875, p = 0.005). With a cut-off value set to 1% intermediate monocytes of leukocytes, this parameter showed a predictive sensitivity and specificity of 75% and 88%. Anti-angiogenic therapy with bevacizumab had no impact on monocyte populations including TEMs. In 15 patients and six healthy controls, the gene expression profile and the migratory behavior of monocyte subsets was evaluated. The profile of intermediate monocytes suggested functions in antigen presentation, inflammatory cytokine production, chemotaxis and was remarkably stable during chemotherapy. Intermediate monocytes showed a preferential migratory response to tumor-derived signals in vitro and correlated with the level of CD14+/CD16+ monocytic infiltrates in the resected tumor tissue. In conclusion, the rapid rise of intermediate monocytes during chemotherapy may offer a simple marker for response prediction and a timely change in regimen.