Multicentre randomized controlled trial comparing ferric(III)carboxymaltose infusion with oral iron supplementation in the treatment of preoperative anaemia in colorectal cancer patients.

BACKGROUND: At least a third of patients with a colorectal carcinoma who are candidate for surgery, are anaemic preoperatively. Preoperative anaemia is associated with increased morbidity and mortality. In general practice, little attention is paid to these anaemic patients. Some will have oral iron prescribed others not. The waiting period prior to elective colorectal surgery could be used to optimize a patients' physiological status. The aim of this study is to determine the efficacy of preoperative intravenous iron supplementation in comparison with the standard preoperative oral supplementation in anaemic patients with colorectal cancer. METHODS/DESIGN: In this multicentre randomized controlled trial, patients with an M0-staged colorectal carcinoma who are scheduled for curative resection and with a proven iron deficiency anaemia are eligible for inclusion. Main exclusion criteria are palliative surgery, metastatic disease, neoadjuvant chemoradiotherapy (5 × 5 Gy = no exclusion) and the use of Recombinant Human Erythropoietin within three months before inclusion or a blood transfusion within a month before inclusion. Primary endpoint is the percentage of patients that achieve normalisation of the haemoglobin level between the start of the treatment and the day of admission for surgery. This study is a superiority trial, hypothesizing a greater proportion of patients achieving the primary endpoint in favour of iron infusion compared to oral supplementation. A total of 198 patients will be randomized to either ferric(III)carboxymaltose infusion in the intervention arm or ferrofumarate in the control arm. This study will be performed in ten centres nationwide and one centre in Ireland. DISCUSSION: This is the first randomized controlled trial to determine the efficacy of preoperative iron supplementation in exclusively anaemic patients with a colorectal carcinoma. Our trial hypotheses a more profound haemoglobin increase with intravenous iron which may contribute to a superior optimisation of the patient's condition and possibly a decrease in postoperative morbidity. TRIAL REGISTRATION: ClincalTrials.gov: NCT02243735 .

Toxicity and complications of preoperative chemoradiotherapy for locally advanced rectal cancer.

BACKGROUND: Capecitabine is an attractive radiosensitizer. In this study acute toxicity and surgical complications were evaluated in patients with locally advanced rectal cancer following total mesorectal excision (TME) after preoperative chemoradiotherapy (CRT) with capecitabine. METHODS: Between 2004 and 2008, consecutive patients with clinical tumour category (cT) 3-4 (with a threatened circumferential resection margin or cT3 within 5 cm of the anal verge) or clinical node category 2 rectal cancer were treated with preoperative CRT (25 × 2 Gy, capecitabine 825 mg/m(2) twice daily, days 1-33). TME followed 6 weeks later. Toxicity was scored according to the Common Terminology Criteria (version 3.0) and Radiation Therapy Oncology Group scoring systems. Treatment-related surgical complications were evaluated for up to 30 days after discharge from hospital using the modified Clavien-Dindo classification. RESULTS: Some 147 patients were analysed. The mean cumulative dose of capecitabine was 95 per cent and 98·0 per cent of patients received at least 45 Gy. One patient died from sepsis following haematological toxicity. Grade 3-5 toxicity developed in 32 patients (21·8 per cent), especially diarrhoea (10·2 per cent) and radiation dermatitis (11·6 per cent). There were no deaths within 30 days after surgery. Anastomotic leakage and perineal wound complications developed after 13 of 47 low anterior resections and 23 of 62 abdominoperineal resections. Surgical reintervention was required in 30 patients. Twenty-seven patients (19·6 per cent) of 138 patients who had a laparotomy were readmitted within 30 days after initial hospital discharge. CONCLUSION: Preoperative CRT with capecitabine is associated with acceptable acute toxicity, significant surgical morbidity but minimal postoperative mortality.

Isolation of rat and human Kupffer cells by a modified enzymatic assay.

A new rapid method is described for the isolation and purification of rat and human Kupffer cells without the need of liver perfusion techniques. Rat livers or small human liver wedge biopsies obtained peroperatively were incubated with pronase under continuous pH registration. Kupffer cells were subsequently separated from other nonparenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation. Identification of Kupffer cells was achieved on the basis of ultrastructural analyses and immunophenotyping.

Isolation of cytotoxic Kupffer cells by a modified enzymatic assay: a methodological study.

Kupffer cell (KC)-mediated cytotoxicity against tumor cells is of interest, since the liver is a major site of metastatic growth of primary colorectal cancer. KC isolation methods from rat livers, to study the tumoricidal properties of these cells, are based on perfusion of the liver and are therefore not suitable for human KC isolation from liver biopsies. In view of application to isolate KC from small wedge human liver biopsies, we have developed an isolation procedure for rat KC that does not require perfusion techniques. Liver tissue fragments were incubated with pronase with continuous pH registration and neutralization. KC were subsequently separated from other non-parenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation. KC and other non-parenchymal cells were identified by immunophenotyping with a cytoplasmic monoclonal antibody ED1 and by ultrastructural analysis. About 3 x 10(6) KC per gram liver were isolated with a final purity of > 95% without loss of viability. To ensure that functionally competent KC were isolated, we assayed cytotoxicity against CC531 tumor cells in a recent developed cell-mediated MTT assay. Maximum cytotoxicity of KC was approximately 40% at an effector to target ratio of 10. In conclusion our approach seems to be a useful and simple method to isolate KC with good functional properties from rat livers, without the need for perfusion techniques.

Enhanced human Kupffer cell-mediated cytotoxicity after activation of the effector cells and modulation of the target cells by interferon-gamma: a mechanistic study at the cellular level.

In this study we demonstrate enhanced Kupffer cell (KC) cytotoxicity against several colorectal cell lines by activation of KC and by modulation of the targets (SW948, WiDR, HT29, and SW620) with IFN-gamma. We demonstrated that soluble TNF-alpha had no effect on these tumor cells, while cytotoxicity against SW948 and WiDR was blocked by anti-TNF-alpha. Experiments using a transwell system stressed the importance of close intercellular contact for this process. Anti-IL-1 did not inhibit cytotoxicity against SW948. Modulation of HT29, WiDR, and SW948 by IFN-gamma (500 U/ml) induced a significant increase in cytotoxicity. We conclude that cell-associated TNF-alpha may be responsible for KC cytotoxicity against SW948, a process requiring close intercellular contact. WiDR is only partly lysed by a TNF-alpha-dependent mechanism, whereas HT29 is not. Furthermore, IFN-gamma is involved in the regulation of tumor susceptibility.

Enhanced killing capacity of human Kupffer cells after activation with human granulocyte/macrophage-colony-stimulating factor and interferon gamma.

In this study we investigated the effect of the cytokines human granulocyte/macrophage-colony-stimulating Factor (hGM-CSF) and interferon gamma (IFN gamma) on human Kupffer-cell-mediated cytotoxicity against the SW948 colon carcinoma cell line. Kupffer cells were isolated from small liver wedge biopsies, taken from 14 patients who had had abdominal surgery for colon carcinoma or partial hepatectomy. The cells were incubated with hGM-CSF (100 ng/ml), or with IFN gamma (100 U/ml) or with their combination and the percentage cytotoxicity was determined using a recently described modified assay. Additional experiments were performed with tumour-necrosis-factor-alpha (TNF alpha)-sensitive U937 cells as target. The TNF alpha secretion of Kupffer cells was measured and we evaluated the effect of TNF alpha on colon tumour targets. We performed human-Kupffer-cell-mediated cytotoxicity blocking experiments with anti-TNF alpha and used paraformaldehyde-fixed Kupffer cells to demonstrate lysis of TNF alpha-sensitive WEHI-164 cells and of SW948 cells. The overall cytotoxicity against SW948 caused by unactivated Kupffer cells (n = 14), and by Kupffer cells activated with hGM-CSF (n = 14), IFN gamma (n = 6) or their combination (n = 6) was respectively: 19.5 +/- 2.6%, 25.3 +/- 2.9%, 41 +/- 9.4% and 45.6 +/- 8% at E/T = 1 and 28.2 +/- 2.9%, 35.6 +/- 3.2%, 55.6 +/- 9.7% and 62.8% at E/T = 5. All differences were statistically significant (P < 0.05). No growth-promoting activity by hGM-CSF on the SW948 tumour cells was observed. U937 cells were highly susceptible to Kupffer-cell-mediated cytotoxicity. The TNF alpha secretion by human Kupffer cells increased in parallel to their cytotoxicity after incubation with these cytokines. Soluble TNF alpha had only a slight anti-proliferative effect on SW948 cells, while specific anti-TNF alpha blocked Kupffer cell cytotoxicity by up to 80%. Finally, paraformaldehyde-fixed Kupffer cells were able to lyse WEHI-164 and SW948 cells. This indicates that expression of cell-associated TNF alpha is the main cytolytic mechanism of human-Kupffer-cell-mediated cytotoxicity. The implications for the use of hGM-CSF and IFN gamma in vivo are discussed.

Laparoscopic surgery in the treatment of colonic polyps.

BACKGROUND: Benign colonic polyps that are impossible to remove with the aid of the flexible colonoscope because of their size or location must be removed surgically. METHODS: Twenty patients with colonic adenomatous polyps that could not be resected by colonoscopy because of size or difficult location (n = 18) or polyps in combination with diverticulitis (n = 2) underwent polyp removal through a small 'assisted' incision in the abdominal wall using a standard 'dissection-facilitated' laparoscopic approach to the affected colonic segment. RESULTS: In six patients the polyp was removed through a colotomy, in three through a limited resection (two ileocaecal and one limited sigmoid resection) and in 11 through a standard colectomy (four right hemicolectomy, one left hemicolectomy, four sigmoid and two anterior resections) because of suspicion of cancer. In only one patient could the polyp not be found during laparoscopy, resulting in a second conventional surgical intervention. In four patients carcinoma was diagnosed in the specimen. CONCLUSION: Precise preoperative localization of the polyp and the use of dissection-facilitated laparoscopic colonic surgery make laparoscopic removal of benign colonic polyps an alternative to an open procedure.

Isolation and purification of large quantities of fresh human Kupffer cells, which are cytotoxic against colon carcinoma.

A new rapid method is described for the isolation and purification of functional active human Kupffer cells without the need of in situ perfusion techniques. Liver wedge biopsies (3 to 5 g), obtained after laparotomy, were incubated with pronase under continuous pH registration. Human Kupffer cells were subsequently separated from other nonparenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation. Kupffer cells, 1.7 +/- 0.4 x 10(6) per gram liver, were isolated with a purity of 95% +/- 3%. Cell-mediated cytotoxicity of Kupffer cells was assayed against a human colon carcinoma cell line (SW948). Kupffer cell cytotoxicity was 42% +/- 9% (mean +/- SD) at an effector-to-target cell ratio of 10 and significantly increased to 73 +/- 17% (P < .05) after activation of Kupffer cells with interferon-gamma. In conclusion, a reliable and relatively simple method is provided to isolate and purify fresh human Kupffer cells in large yields, which show spontaneous as well as gamma-interferon-induced cytotoxicity against a human colon carcinoma cell line.

Fresh colorectal tumor cells isolated from individual patients differ in their susceptibility to monocyte mediated cytotoxicity.

Studies on monocyte/macrophage mediated cytotoxicity usually pertain to the use of cell lines that are liable to antigenic and structural changes. Therefore we compared monocyte mediated cytotoxicity against colorectal tumor cell lines (WiDR, HT29, SW620, and SW948) with fresh colorectal tumor cells from patients. Fresh tumor cells were isolated from surgical specimens by a short enzymatic treatment (Collagenase/DNAse). Monocytes were obtained from one healthy donor. Cytotoxicity was determined using the MTT-assay. Fresh colorectal tumor cells displayed a similar differential susceptibility to cytotoxic monocytes as cell lines. Cytotoxicity against fresh tumor cells ranged from 4.9% to 50.4% at E/T ratio 5 (n = 9). Activation of monocytes with Interferon-gamma (100 U/ml) induced an increase of 6.2% +/- 1.6 (n = 4, P = 0.06). In this study we demonstrate monocyte mediated cytotoxicity against colorectal tumor cells isolated from individual patients. This may be important in view of the development of adoptive immunotherapy and cell-directed immunotherapy.

Enhanced tumour growth in the rat liver after selective elimination of Kupffer cells.

The evidence that Kupffer cells are capable of controlling metastatic growth in the liver in vivo is largely circumstantial. The best approach when studying natural cytotoxicity activities of Kupffer cells is to investigate the effect of Kupffer cell elimination on tumour growth. Until now it has not been possible to eliminate Kupffer cells without affecting other cell populations. We have recently developed a new method to eliminate Kupffer cells selectively: intravenous injection of liposome-encapsulated (dichloromethylene)bisphosphonate (Cl2MDP-liposomes) leads to effective elimination of all Kupffer cells, without affecting non-phagocytic cells. Wag/Rij rats were injected with Cl2MDP-liposomes. After 48 h, rats were inoculated with syngeneic CC531 colon carcinoma cells by injection in the portal system. The results show a strongly enhanced tumour growth in the liver of the Cl2MDP-liposome-treated rats. In these animals, livers were almost completely replaced by tumour and had increased in weight, whereas in the control groups only a few (four to eight) small (1-mm) tumour nodules were found. These data show that selective elimination of Kupffer cells results in enhanced tumour growth in the liver, implying that Kupffer cells play a crucial role in controlling tumour growth in the liver.

Macrophage populations in different stages of induced hepatic metastases in rats: an immunohistochemical analysis.

Macrophages play a role in the host defence against cancer. Little is known about changes in macrophage populations during early metastatic growth. To evaluate the distribution, number and phenotype of macrophages in the development of hepatic metastases in a rat model (Wag/Rij rats and syngeneic CC531 colon carcinoma cell line), an immunohistochemical study was performed with the monoclonal antibodies ED1 (monocytes, and all macrophages), ED2 (resident tissue macrophages, like Kupffer cells) and ED3 (a subpopulation of macrophages which may play a role in the recruitment of lymphocytes). OX19 and His14 were used to identify lymphocytes. In this study a new monoclonal antibody CC52 is described, which recognizes the CC531 tumour cell line. Liver metastases were induced by injection of CC531 colon carcinoma cells into a mesenteric vein. Rats were killed at various intervals. Results show three major macrophage populations during hepatic tumour growth: (1) on day 3, infiltrates are observed around the micrometastases, which contain mainly newly recruited macrophages (ED1+ and ED2-); (2) after 7 days, ED3-positive (ED3+) macrophages together with T lymphocytes are found in the infiltrates; (3) an increase in the number of ED2-positive (ED2+) Kupffer cells is observed in the liver parenchyma after 14 days. In conclusion, the present results suggest that various populations of macrophages, newly recruited (ED1+) as well as resident Kupffer cells (ED2+), are involved in the immune response against tumour cell deposits in the liver.

The combination 5-fluorouracil/levamisole induces enhanced rat Kupffer cell-mediated cytotoxicity in vitro against the syngeneic colon adenocarcinoma cell line CC531.

The mode of action of the combination treatment 5-fluorouracil (5-FU) and levamisole in colorectal cancer patients is unknown. It is postulated that the beneficial effect may be explained by an immunomodulatory effect on Kupffer cell (KC) cytotoxicity. We evaluated the effect of levamisole (200 micrograms/ml) and 5-FU (10 microM) on rat KC cytotoxicity against syngeneic CC531 tumor cells. Viability of KCs was unaffected by 5-FU and/or levamisole. The combination did not enhance growth inhibition of CC531 compared to 5-FU alone. A significant increase in KC cytotoxicity was observed after 24-hr incubation with 5-FU/levamisole especially at an effector/target ratio of 10 (P < 0.05). 5-FU alone had no effect on KC cytotoxicity, while levamisole induced only a slight increase. Our in vitro data suggest that the additive effect of the combination 5-FU/levamisole on KC cytotoxicity may attribute to the beneficial effect of the adjuvant treatment in colorectal cancer patients.