Hubert G. Hotz

Risk factors for clinical anastomotic leakage and postoperative mortality in elective surgery for rectal cancer

Background and aimsClinical anastomotic leakage remains a major problem after anterior or low anterior resection for rectal cancer. The aim of this study was to assess the association between risk factors and anastomotic leakage and postoperative mortality.Materials and methodsTwo hundred seventy-six elective anterior or low anterior resections with anastomosis were performed and documented on-line from January 1995 to December 2004. Univariate and multivariate analyses with Bonferroni adjustment were carried out to identify relevant risk factors.ResultsThe rate of anastomotic leakage was 14.9% (41 of 276 patients) with a mortality of 12.2% (5 of 41 patients). Overall mortality was 2.5% (7 of 276 patients). Multiple regression analysis showed that smokers had an increased risk of anastomotic leakage [odds ratio (OR), 6.42; 95% confidence interval (CI), 2.68–15.36] as well as patients with coronary heart disease (OR, 7.79; 95% CI, 2.52–24.08). Smokers (OR, 13.20; 95% CI, 2.48–7.24) and patients with coronary heart disease (OR, 23.46; 95% CI, 4.33–27.04) also had an increased risk of postoperative mortality in the univariate analysis as well as patients with anastomotic leakage (OR, 16.25; 95% CI, 3.04–16.92).ConclusionsSmoking and coronary heart disease are important risk factors for anastomotic leakage and postoperative mortality after elective resection for rectal cancer.

Beyond Epithelial to Mesenchymal Transition: A Novel Role for the Transcription Factor Snail in Inflammation and Wound Healing

IntroductionSnail, a transcription factor linked to epithelial to mesenchymal transition (EMT) during embryonic development and tumor progression, is associated with migration of cells. During inflammation and tissue injury, cell movement is also observed to provide the first line of defense against bacteria and to promote wound healing. Therefore, we studied the function of Snail in activated macrophages in a variety of inflammatory processes.Materials and MethodsIn this study, we examined the expression and localization of Snail during inflammation and tissue injury in rats and human tissue specimens, by immunohistochemistry, Western blot, and real-time PCR. We investigated Snail expression after stimulation of macrophages with TGF-β1, LPS, Interleukin-8, and MMP-3 in vitro. To further understand the role of Snail in activated macrophages, we used Stealth siRNA against Snail, transfected the human macrophage cell line THP-1, and measured migration of cells in an in vitro invasion assay.Results and DiscussionWe found a strong, transient, and time-dependent activation of Snail in migrating macrophages at the sites of injury in vivo and in vitro, as well as in patients with inflammatory bowel disease. Furthermore, we showed that induction of Snail in macrophages is dependent on TGF-β1 signaling pathway. Downregulation of Snail by Stealth siRNA led to impaired migration of THP-1 cells in an invasion assay after stimulation with TGF-β1.ConclusionWe conclude that TGF-β1 induced migration of activated macrophages during inflammation and wound healing is mediated by snail. These results give insights in a novel EMT-like mechanism present in immune cell movement during tissue injury.

In vitro and in vivo antitumor activity of cetuximab in human gastric cancer cell lines in relation to epidermal growth factor receptor (EGFR) expression and mutational phenotype

BackgroundTargeting the epidermal growth factor receptor (EGFR) pathway is an important approach for a variety of tumors. This study assessed the effect of cetuximab, an anti-EGFR monoclonal antibody, on three gastric cancer cell lines with different phenotypes in vitro and in a therapeutic orthotopic murine gastric cancer model.MethodsThree human gastric cancer cell lines (AGS, MKN-45, NCI-N87) were evaluated for cell surface EGFR expression, and K-ras and BRAF mutations. In vitro, the effects of cetuximab, carboplatin, irinotecan, and docetaxel were investigated. Orthotopic tumors derived from MKN-45 and NCI-N87 were established in nude mice. After 4xa0weeks, the animals received cetuximab (1xa0mg/kg, weekly i.p.) or carboplatin (20xa0mg/kg, weekly i.p.), or both agents. The volume of the primary tumor and local and systemic tumor spread were determined at autopsy at 14xa0weeks. Tumor sections were immunostained for EGFR, as well as stained for CD31 to analyze microvessel density.ResultsCell surface expression of EGFR was found only in AGS and NCI-N87 cells. AGS cells displayed a codon 12 K-ras mutation, and all three cell lines were BRAF wild-type. In vitro, cetuximab significantly reduced cell viability and proliferation only in EGFR-positive/K-ras wild-type NCI-N87 cells (−48%). In vivo, cetuximab in combination with carboplatin synergistically reduced tumor volume (−75%), dissemination (−63%), and vascularization (−47%) in NCI-N87 xenografts. Tumors derived from EGFR-negative MKN-45 cells were unaffected by cetuximab.ConclusionsCetuximab is effective in K-ras wild-type, EGFR-expressing gastric cancer cell lines and xenografts. In vivo, the combination of cetuximab with carboplatin displayed synergistic antitumor activity.

Suramin inhibits not only tumor growth and metastasis but also angiogenesis in experimental pancreatic cancer.

Suramin inhibits the proliferation of several human tumors in vivo and in vitro. In this study, the effects of Suramin on proliferation and angiogenesis were investigated in human pancreatic cancer cell lines and in an orthotopic nude mouse model of human pancreatic cancer. The effects of Suramin on proliferation, viability, cell cycle, and apoptosis were studied in five human pancreatic cancer cell lines. Suramin inhibited the proliferation of pancreatic cancer cells in a dose-dependent manner and reduced viability at high concentrations. Cell cycle analysis revealed a decreased S-phase fraction in most cell lines, whereas the apoptotic fraction was not notably different. In vivo treatment with Suramin significantly reduced pancreatic tumor size (MiaPaCa-2, −74%; AsPC-1, −41%; and Capan-1, −49%) and metastatic spread (MiaPaCa-2, −79%; AsPC-1, −34%; and Capan, −38%). As a parameter for angiogenic activity, vascular endothelial growth factor (VEGF) secretion was measured, revealing reduced VEGF concentrations under Suramin treatment in both cell culture medium and ascites. Also, microvessel density quantified in primary tumors was reduced in animals treated with Suramin. Therefore, Suramin inhibits the proliferation of human pancreatic cancer in vitro and in vivo. The therapeutic effects seem to involve cell cycle kinetics and may be in part related to the antiangiogenic action of the drug.

Effect of Anastomosis Level on Continence Performance and Quality of Life after Colonic J-pouch Reconstruction

Total mesorectal excision (TME) has become the recommended method for treatment of cancer in the middle or lower third of the rectum. Thus very low anastomoses are necessary to preserve continence, and pouch reconstruction is favored. It is unclear whether the level of anastomosis is important for continence and quality of life in colonic J-pouch reconstruction. In this investigation all patients were included who underwent curative elective anterior continuity resection with colorectal or coloanal J-pouch reconstruction for primary rectal cancer between January 2001 and December 2004. Exclusion criteria were distant metastases and any signs of recurrence at the time of investigation. Evaluation of continence performance by Wexner and Holschneider questionnaire and quality of life using the QLQ-C30 and QLQ-CR38 (EORTC) questionnaires was done 220xa0±xa038xa0days after closure of the protective Ileostomy, which was performed 106xa0±xa048xa0days after primary intervention. Fifty-two patients (79%) were analyzed. Colopouch rectal anastomosis was performed in eighteen cases and colopouch anal anastomosis in thirty-four cases. Fifty percent of the patients in both groups were continent for solid stool. Patients with a colopouch anal anastomosis had a significantly higher rate of incontinence for liquid stool, however. They took stool-regulating medicine more frequently and complained of fecal soiling and a restricted quality of life. Patients with a colopouch anal anastomosis had a significantly lower score on the most important points of the QLQ-C30 (emotional functioning, social functioning, pain, and quality of life). The same applied to the QLQ-CR38 for body image and problems with defecation. The quality of life of patients with a colopouch anal anastomosis was still considered acceptable compared with reference data for the normal healthy population, however. Both continence and quality of life are substantially affected by the level of the anastomosis after colonic pouch reconstruction. This suggests preservation of a small part of the rectum when oncologically feasible and performing a colopouch rectal anastomosis.

Long-term outcome of “prophylactic therapy” for familial medullary thyroid cancer

BACKGROUNDnAbout one quarter of all medullary thyroid cancers (MTC) are determined genetically due to a mutation in the RET proto-oncogene. The most common site of mutation is in codon 634. Therapeutic approaches toward patients at risk for the development of MTC identified by family screening programs range from total thyroidectomy to total thyroidectomy with lymphadenectomy of all 4 compartments.nnnMETHODSnWe report 17 patients (median age, 13 years; range, 4-36) carrying a mutation in codon 634 of the RET proto-oncogene who were operated on prophylactically at our department. All patients underwent thyroidectomy with bilateral cervicocentral lymphadenectomy. Current calcitonin level, overall survival, and disease-free survival were analyzed by contacting general practitioners and patients.nnnRESULTSnTumor classification was as follows: C-cell hyperplasia, 18% (n = 3); T1 (<1 cm), 71% (n = 12); and T1 (>1 cm), 12% (n = 2). Only 2 patients had lymph node metastases (12%). These patients developed recurrent disease (median observation time, 147 months; range, 90-181). In 1 patient, the calcitonin level normalized after unilateral cervicolateral lymphadenectomy. The other patient (9 years old at primary operation) still has a persistently increased serum calcitonin level after 140 months of follow-up despite several operations for MTC.nnnCONCLUSIONnTotal thyroidectomy with bilateral cervicocentral lymphadenectomy is sufficient as routine prophylactic therapy for patients with mutations in codon 634 of the RET proto-oncogene. Cervicolateral lymphadenectomy is indicated if calcitonin remains elevated after primary surgery. Prophylactic thyroidectomy should be performed before the development of lymph node metastases.

Selective inhibition of endothelin receptor A as an anti-angiogenic and anti-proliferative strategy for human pancreatic cancer

Endothelin-1 (ET-1) plays a major role in tumor proliferation and angiogenesis of various types of cancer acting through endothelin receptors A and B (ETRA and ETRB). The aim of this study was to analyze theET-1/ETRsystem inhumanpancreatic cancer cell linesandto evaluate the effect of a selective endothelin A inhibitor in vitro and in vivo in an orthotopic mouse model. Three different human pancreatic cancer cell lines, MiaPaCa-2, AsPC-1, and Panc-1, were studied. We found that proliferation of human pancreatic carcinoma cells expressing ETRA was significantly reduced with a selective antagonist. Hypoxic conditions led to improved results compared to a normoxic environment (MiaPaCa-2: -53% vs. -18%; AsPC-1: -54% vs. -46%). Proliferation of ETRA negative Panc-1 cells was not decreased. In vivo, the selective ETRA inhibition resulted in reduced angiogenesis as measured by lower microvessel densities (MiaPaCa-2: -47%; AsPC-1: -55%). The blockade of ETRA decreased the volume (MiaPaCa-2: -87%; AsPC-1: -28%) and metastatic spread (MiaPaCa-2: -95.5%; AsPC-1: -27%) of receptorpositive tumors, thereby increasing survival in experimental pancreatic cancer. ETRA blockade did not show an effect on ETRA negative Panc-1 tumors. Therefore, targeting ETRA with a selective antagonist might provide a new approach to reducing proliferation and angiogenesis in human pancreatic cancer.

An orthotopic nude mouse model for preclinical research of gastric cardia cancer

PurposeA clinically relevant animal model for cancer of the esophagogastric junction does not exist. This study aimed to establish an orthotopic mouse model for human gastric cancer of the distal stomach and the gastric cardia.Materials and methodsHuman gastric cancer cell lines AGS, MKN-45, and NCI-N87 were injected subcutaneously into nude mice. These donor tumors were harvested after 4xa0weeks and minced into small tumor fragments. One donor tumor fragment was orthotopically implanted into the submucosa of either gastric cardia or distal stomach in other mice. The animals were killed 4, 8, and 12xa0weeks after tumor implantation. Volume of the primary tumor and local and systemic tumor spread were determined.ResultsThe implantation technique resulted in a tumor take rate of 100%. An artificial dissemination of tumor cells into the abdominal cavity due to the procedure was not observed.ConclusionsWe report for the first time the development of a clinically relevant mouse model for human gastric cancer of the gastric cardia and the distal stomach. Primary tumor growth and local and systemic spread progressed continuously during the observation period and mimic the human situation of this disease. This model may be suitable to evaluate novel treatment strategies for this malignancy.

Fluid resuscitation with human albumin or hydroxyethyl starch--are there differences in the healing of experimental intestinal anastomoses?

Abstract Objectives. Restoration of the macro- and microcirculation is important for the healing of gastrointestinal anastomoses. Colloids and crystalloids are widely used for blood volume therapy. We evaluated the effects of human albumin, hydroxyethyl starch (HES) 130/0.4 and saline on the microcirculation and on wound healing in colon anastomoses in rats. Material and methods. Male Wistar rats received a colonic end-to-end anastomosis. The animals were randomized into three groups and a single 3-ml dose of either 20% human albumin, 6% HES 130/0.4 or 0.9% saline was applied intravenously. Six, 24, 48, 96 h and 2 weeks after the procedure, 10 animals per group were reanesthetized. Measurements of capillary blood flow, vessel permeability and anastomosis bursting pressure were performed. The amounts of vascular endothelial growth factor (VEGF) and IL-6 in the plasma were determined by enzyme-linked immunosorbent assays, and the mRNA levels of VEGF and collagen types I and III were measured by real-time polymerase chain reaction. Results. No significant differences were found between albumin, HES 130/0.4 and saline in capillary blood flow, vessel permeability and anastomotic bursting pressure in this rat model. Concentrations of collagen I and III mRNA were significantly elevated after 96 h in animals that had received HES 130/0.4 or albumin. RNA and protein levels of VEGF and interleukin-6 were unaffected by therapy. Conclusions. Human albumin, which is still widely used in the clinical setting, had no advantage over HES 130/0.4 and saline with regard to anastomotic healing in this animal model. Nevertheless, we prefer HES 130/0.4 because it is more effective for volume therapy than saline and has a better availability and is less expensive than human albumin.

Dendritic cells from human mesenteric lymph nodes in inflammatory and non-inflammatory bowel diseases: subsets and function of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDC) in mesenteric lymph nodes (MLN) may be important regulators of both inflammatory and non‐inflammatory mucosal immune responses but human studies are rare. Here we compare pDC from human MLN and peripheral blood (PB) by phenotype and function. MLN from patients with or without inflammatory bowel disease (IBD) undergoing colon surgery and PB from patients with IBD and from controls were used to isolate mononuclear cells. The pDC were analysed by flow cytometry for the expression of CD40, CD80, CD83, CD86, CCR6, CCR7, CX3CR1, CD103 and HLA‐DR. Purified pDC from MLN and PB were stimulated with staphylococcus enterotoxin B (SEB), CpG‐A, interleukin‐3 (IL‐3), SEB + IL‐3, CpG‐A + IL‐3 or left unstimulated, and cultured alone or with purified allogeneic CD4+ CD45RA+ HLA‐DR‐ T cells. Subsequently, concentrations of IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12, IL‐17, interferon‐α (IFN‐α), IFN‐γ and tumour necrosis factor‐α (TNF‐α) in culture supernatants were determined by multiplex bead array. The PB pDC from IBD patients exhibited an activated and matured phenotype whereas MLN pDC and control PB pDC were less activated. CpG‐A and CpG‐A + IL‐3‐stimulated MLN pDC secreted less IL‐6 and TNF‐α compared with PB pDC from controls. Compared with co‐cultures of naive CD4 T cells with PB pDC, co‐cultures with MLN pDC contained more IL‐2, IL‐10 and IFN‐γ when stimulated with SEB and SEB + IL‐3, and less IFN‐α when stimulated with CpG‐A. MLN pDC differ phenotypically from PB pDC and their pattern of cytokine secretion and may contribute to specific outcomes of mucosal immune reactions.