Lejla Alidzanovic

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.

Thrombocytes Correlate with Lymphangiogenesis in Human Esophageal Cancer and Mediate Growth of Lymphatic Endothelial Cells In Vitro.

Recent data provide evidence for an important role of thrombocytes in lymphangiogenesis within human malignant disease. The aim of this study was to investigate the role of thrombocytes in lymphangiogenesis in human esophageal cancer. Perioperative peripheral blood platelet counts (PBPC) were evaluated retrospectively in 320 patients with esophageal cancer, comprising 184 adenocarcinomas (AC), and 136 squamous cell carcinomas (SCC). Data on lymphangiogenesis evaluated by anti-podoplanin immunostaining were available from previous studies, platelets within the tumor tissue were assessed by CD61 immunostaining. For in vitro studies, human lymphatic endothelial cells (LECs) were isolated and co-cultured with peripheral blood platelets. Stromal thrombocytic clusters (STC) were evident in 82 samples (25.6%), and vascular thrombocytic clusters (VTC) in 56 (17.5%). STC and VTC were associated with a significantly higher PBPC at investigation of all cases. The presence of STC was associated with higher lymphatic microvessel density (p<0.001), PBPC and STC were associated with lymphovascular invasion of tumor cells in a regression model. The presence of STCs was associated with shorter DFS of all patients (p = 0.036, Breslow test), and VTC with shorter DFS in in SCC (p = 0.025, Breslow test). In cell culture, LEC proliferation was enhanced by co-culture with human platelets in a dose- and time-dependent manner mediated by the release of PDGF-BB and VEGF-C. Platelets play an important role in lymphangiogenesis and lymphovascular invasion in esophageal cancer, influencing prognosis. So the disruption of signaling pathways between platelets, tumor cells and lymphatic endothelium might be of benefit for patients.

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.

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.

Activity of bevacizumab at the time of surgery after 6 weeks of preoperative cessation.

455 Background: The use of bevacizumab (BV), a monoclonal antibody against VEGF, and combination chemotherapy (CTx) together with an increase in hepatic resection has improved the outcome of colorectal cancer (CRC) patients with liver metastasis. However, despite the frequent use of BV, its accurate timing prior to liver resection to avoid surgical complications and to allow for hepatic regeneration remains discussed controversially. In this context, it remains unclear to which extent VEGF is complexed by BV at the time of surgery. METHODS Fifty CRC patients with liver metastases, potentially curable by resection, were included. Patients received neoadjuvant CTx either with or without concomitant biweekly BV. The last cycle of therapy did not include BV, resulting in 5 weeks between the last administration of BV and surgery. Plasma and wound fluid subcutaneously and intra-abdominally were obtained perioperatively. Immunoprecipitation was applied to determine the proportion of BV-bound VEGF. RESULTS 42 patients underwent surgery. Median time of BV cessation was 5-6 weeks. No increased perioperative complications in this BV treated patients were observed (patients without complications: 59 % of BV + CTx patients versus 50 % in CTx patients). At the time of surgery, the majority of plasma VEGF was inactivated by BV. In wound fluid, the actual site of wound healing and hepatic regeneration, VEGF was remarkably low and also complexed by BV. This resulted in a highly significant reduction of unbound/biologically active VEGF levels in plasma and wound fluid of BV treated patients (both: P < 0.001). CONCLUSIONS These data documents that, after the clinical standard cessation time point of 5-6 weeks, BV is still fully active and binds to the majority of circulating and local (wound fluid) VEGF. Despite the potent VEGF inactivation, at the time of liver resection no substantial increase in perioperative morbidity after neoadjuvant BV therapy has been documented in our study and the current literature.

Association of VEGF feedback production in bevacizumab therapy with systemic response.

3056 Background: The monoclonal antibody bevacizumab (Beva) has proven a promising tool for anti-angiogenic cancer therapy by neutralizing vascular endothelial growth factor A (VEGF). However, in response to Beva administration an increased level of VEGF has been detected in patient blood. Although this feedback mechanism has been observed in several studies, it remains unclear if VEGF production is caused by the tumor itself or reflects a systemic host response. Furthermore, indirect effects of VEGF neutralization on other angiogenic mediators are of interest, as they may lead to compensation or therapy resistance. Under these aspects we have monitored levels of VEGF, circulating endothelial cells (CECs) and Tie-2 expressing monocytes (TEMs) in the context of a clinical trial. Methods: 18 patients with stage IV colorectal cancer and resection of liver metastases have been investigated to date. Blood samples were taken at the beginning and the end of their neoadjuvant as well as adjuvant treatment based o...