Versican and vascular endothelial growth factor expression levels in peritoneal metastases from colorectal cancer are associated with survival after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) can increase survival of colorectal cancer (CRC) patients with peritoneal metastases (PM). This treatment is associated with high morbidity and mortality rates. Therefore, improvement of patient selection is necessary. Assuming that the clinical phenotype is dictated by biological mechanisms, biomarkers could play a crucial role in this process. Since it is unknown whether and to what extent angiogenesis influences the course of disease in patients with PM, we investigated the expression of two angiogenesis-related markers and their relation to overall survival (OS) in CRC patients after CRS and HIPEC. Clinicopathological data and tissue samples were collected from 65 CRC patients with isolated metastases to the peritoneum that underwent CRS and HIPEC. Whole tissue specimens from PM were evaluated for versican (VCAN) expression, VEGF expression and microvessel density (MVD) by immunohistochemistry. The relation between these markers and OS was assessed using univariate and multivariate analysis. Associations between VEGF expression, VCAN expression, MVD and clinicopathological data were tested. High stromal VCAN expression was associated with high MVD (p = 0.001), better resection outcome (p = 0.003) and high T-stage (p = 0.027). High epithelial VCAN expression was associated with MVD (p = 0.007) and a more complete resection (p < 0.001). In multivariate analysis, simplified peritoneal cancer index (p = 0.001), VEGF expression levels (p = 0.012), age (p = 0.030), epithelial VCAN expression levels (p = 0.042) and lymph node status (p = 0.053) were associated with OS. Concluding, VCAN and VEGF were associated with survival in CRC patients with PM after CRS and HIPEC. Independent validation in a well-defined patient cohort is required to confirm the putative prognostic role of these candidate biomarkers.

Angiogenesis-Related Markers and Prognosis After Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy for Metastatic Colorectal Cancer.

BACKGROUND: Patients presenting with peritoneal metastases (PM) of colorectal cancer (CRC) can be curatively treated with cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). Angiogenesis is under control of multiple molecules of which HIF1a, SDF1, CXCR4, and VEGF are key players. We investigated these angiogenesis-related markers and their prognostic value in patients with PM arising from CRC treated with CRS and HIPEC. PATIENTS AND METHODS: Clinicopathological data and tissue specimens were collected in 2 tertiary referral centers from 52 patients who underwent treatment for isolated PM of CRC. Whole tissue specimens were subsequently analyzed for protein expression of HIF1a, SDF1, CXCR4, and VEGF by immunohistochemistry. Microvessel density (MVD) was analyzed by CD31 immunohistochemistry. The relationship between overall survival (OS) and protein expression as well as other clinicopathological characteristics was analyzed. RESULTS: Univariate analysis showed that high peritoneal cancer index (PCI), resection with residual disease and high expression of VEGF were negatively correlated with OS after treatment with CRS and HIPEC (P < 0.01, P < 0.01, and P = 0.02, respectively). However, no association was found between the other markers and OS (P > 0.05). Multivariate analysis showed an independent association between OS and PCI, resection outcome and VEGF expression (multivariate HR: 6.1, 7.8 and 3.8, respectively, P ≤ 0.05). CONCLUSIONS: An independent association was found between high VEGF expression levels and worse OS after CRS and HIPEC. The addition of VEGF expression to the routine clinicopathological workup could help to identify patients at risk for early treatment failure. Furthermore, VEGF may be a potential target for adjuvant treatment in these patients.

Tailoring heated intraperitoneal mitomycin C for peritoneal metastases originating from colorectal carcinoma: a translational approach to improve survival.

BACKGROUND: Patients with peritoneal metastases (PMs) originating from colorectal carcinoma (CRC) are curatively treated by cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) with mitomycin C (MMC). We aim to improve patient selection for HIPEC by predicting MMC sensitivity. METHODS: The MMC sensitivity was determined for 12 CRC cell lines and correlated to mRNA expression of 37 genes related to the Fanconi anaemia (FA)-BRCA pathway, ATM-ATR pathway and enzymatic activation of MMC. Functionality of the FA-BRCA pathway in cell lines was assessed using a chromosomal breakage assay and western blot for key protein FANCD2. Bloom syndrome protein (BLM) was further analysed by staining for the corresponding protein with immunohistochemistry (IHC) on both CRC cell lines (n=12) and patient material (n=20). RESULTS: High sensitivity correlated with a low BLM (P=0.01) and BRCA2 (P=0.02) at mRNA expression level. However, FA-BRCA pathway functionality demonstrated no correlation to MMC sensitivity. In cell lines, weak intensity staining of BLM by IHC correlated to high sensitivity (P=0.04) to MMC. Low BLM protein expression was significantly associated with an improved survival in patients after CRS and HIPEC (P=0.04). CONCLUSIONS: Low BLM levels are associated with high MMC sensitivity and an improved survival after HIPEC.

Cytoreductive surgery and HIPEC for peritoneal metastases combined with curative treatment of colorectal liver metastases: Systematic review of all literature and meta-analysis of observational studies.

OBJECTIVE: Assess the overall outcome in colorectal cancer (CRC) patients that present with a combination of peritoneal metastases (PM) and liver metastases (CRLM) after curative resection and hyperthermic intraperitoneal chemotherapy (HIPEC) in the current literature. METHODS: A systematic literature search according to the PRISMA guidelines was conducted using the PubMed database of the U.S. National library of Medicine using the keywords: colorectal cancer, liver metastasis, extra-hepatic, peritoneal metastases, peritoneal carcinomatosis, cytoreductive surgery (CRS), HIPEC and combinations hereof. Papers focussing on CRS and HIPEC for PM combined with curative treatment of CRLM were included, provided sufficient information on survival outcomes could be extracted. Duplicate publications were excluded. Meta-analysis was performed using the method described by Tierney et al. RESULTS: After screening and full-text assessment of 39 papers, six articles were included containing data on combined PM and CRLM in patients treated with curative resection of both sites and HIPEC or early postoperative intraperitoneal chemotherapy (EPIC). Three articles provided enough statistical information for meta-analysis. Pooled hazard ratio (HR) was extracted from survival curves and was 1.24 (CI 0.96-1.60). A comparison was made with patients presenting with isolated PM undergoing CRS and HIPEC and with patients with disseminated disease undergoing (modern) systemic chemotherapy. CONCLUSIONS: In the absence of randomized controlled studies, we found in this systematic review and meta-analysis of patients with a combination of colorectal metastases in the liver as well as in the peritoneum show a trend towards a lower overall survival after curative resection and HIPEC, when compared to patients with isolated peritoneal metastases after CRS and HIPEC (pooled HR1.24, CI 0.96-1.60). However, patients with metastatic CRC show a tendency towards increased median overall survival after CRS and HIPEC combined with resection of liver metastases when compared to treatment with modern systemic chemotherapy.

Understanding molecular mechanisms in peritoneal dissemination of colorectal cancer : future possibilities for personalised treatment by use of biomarkers.

When colorectal cancer (CRC) metastasizes, this is mostly to the liver via the portal circulation. In addition, 10-25 % of CRC patients eventually show metastases in the peritoneum. A selection of these patients is treated with cytoreductive surgery (CRS) and hyperthermic intra-peritoneal chemotherapy (HIPEC). However, several clinical needs still exist in which biomarkers could play an important role. Relatively little is known about the biology of peritoneal spread of CRC. The development of peritoneal metastases (PM) involves several steps, including: detachment of malignant cells; anoikis evasion; attachment to and invasion of the peritoneal surface ultimately ending in a colonization phase in which the malignant cells thrive in the newly formed niche. In this paper, we provide an overview of molecules associated with peritoneal dissemination and explore the clinical possibilities of these candidate biomarkers. A literature search was conducted using the PubMed database of the U.S. National Library of Medicine and Medline to identify studies on the biological behaviour of PM of CRC. In a series of over 100 studies on PM published between 1990 and 2010, IGF-1, HIF1α, VEGF, EGFR and ITGB1 emerge as the most interesting candidates for possible clinical application. Even though these promising candidate biomarkers have been identified, all of these require extensive further validation prior to clinical application. Yet, the pace of the omics revolution makes that the question is not if, but when biomarkers will be introduced to improve diagnosis and ultimately outcome of patients with PM due to CRC.

High lymph node yield is related to microsatellite instability in colon cancer.

BACKGROUND: Lymph node (LN) yield in colon cancer resection specimens is an important indicator of treatment quality and has especially in early-stage patients therapeutic implications. However, underlying disease mechanisms, such as microsatellite instability (MSI), may also influence LN yield, as MSI tumors are known to exhibit more prominent lymphocytic antitumor reactions. The aim of the present study was to investigate the association of LN yield, MSI status, and recurrence rate in colon cancer. METHODS: Clinicopathological data and tumor samples were collected from 332 stage II and III colon cancer patients. DNA was isolated and PCR-based MSI analysis performed. LN yield was defined as "high" when 10 or more LNs were retrieved and "low" in case of fewer than 10 LNs. RESULTS: Tumors with high LN yield were significantly associated with the MSI phenotype (high LN yield: 26.3% MSI tumors vs low LN yield: 15.1% MSI tumors; P=.01), mainly in stage III disease. Stage II patients with high LN yield had a lower recurrence rate compared with those with low LN yield. Patients with MSI tumors tended to develop fewer recurrences compared with those with MSS tumors, mainly in stage II disease. CONCLUSIONS: In the present study, high LN yield was associated with MSI tumors, mainly in stage III patients. Besides adequate surgery and pathology, high LN yield is possibly a feature caused by biologic behavior of MSI tumors.

The use of fluorescent dyes and probes in surgical oncology.

AIMS AND BACKGROUND: Improved visualization of surgical targets inside of the patient helps to improve radical resection of the tumor while sparing healthy surrounding tissue. In order to achieve an image, optical contrast must be generated by properties intrinsic to the tissue, or require the attachment of special visualization labels to the tumor. In this overview the current status of the clinical use of fluorescent dyes and probes are reviewed. METHODS: In this review, all experimental and clinical studies concerning fluorescent imaging were included. In addition, in the search for the optimal fluorescent imaging modality, all characteristics of a fluorescent dye were described. FINDINGS AND CONCLUSIONS: Although the technique of imaging through fluorescence sounds promising and several animal models show efficacy, official approval of these agents for further clinical evaluation, is eagerly awaited.

HER-family gene amplification and expression in resected pancreatic cancer.

AIMS: Despite surgical resection, pancreatic cancer carries a poor prognosis. In search for new molecular therapeutic targets, we investigated the expression of the HER-family and gene amplification of HER-2 in pancreatic adenocarcinomas of different stages. METHODS: Tissue of 45 resected patients was analyzed for all HER-family 1-4 expression by immunohistochemistry and HER-2 gene amplification was assessed by multiplex ligation-dependent probe amplification and chromogenic in situ hybridization. The type of surgery, location, stage and grade of the tumor, as well as involvement of the resection margins were correlated with HER-expressions and univariate and multivariate survival analysis performed. RESULTS: Normal pancreatic tissue lacked HER1-2 expression, but did show HER3-4 expression. In cancers, no membranous overexpression of HER-1 and HER-2 was seen nor gene amplification of HER-2 found. HER-3, HER-4 is physiologically expressed in the normal pancreas and loss of cytoplasmic HER-3 and HER-4 expression was seen in 33/45 (73%) and 8/45 (18%) of pancreatic cancers. Cytoplasmic HER-3 expression decreased from early to late stage (p=0.05). HER-4 expression was not associated with survival, stage or tumor grade. There were no statistically significant differences in HER1-4 expression between the papilla of Vater (n=13) and non-papilla cancers (n=32). Multivariate survival analysis showed only stage to be of independent prognostic value (p=0.015). CONCLUSIONS: HER-1 and HER-2 are not overexpressed in pancreatic cancers. HER-3 and HER-4 are expressed in the normal pancreas but expression is lost in pancreatic cancer. HER-targeted therapy in pancreatic cancer is not supported by HER-expression of the tumor.

Minimally invasive pediatric surgery: Increasing implementation in daily practice and resident's training.

BACKGROUND: In 1998, the one-year experience in minimally invasive abdominal surgery in children at a pediatric training center was assessed. Seven years later, we determined the current status of pediatric minimally invasive surgery in daily practice and surgical training. METHODS: A retrospective review was undertaken of all children with intra-abdominal operations performed between 1 January 2005 and 31 December 2005. RESULTS: The type of operations performed ranged from common interventions to demanding laparoscopic procedures. 81% of all abdominal procedures were performed laparoscopically, with a complication rate stable at 6.9%, and conversion rate decreasing from 10% to 7.4%, compared to 1998. There were six new advanced laparoscopic procedures performed in 2005 as compared to 1998. The children in the open operated group were significantly smaller and younger than in the laparoscopic group (p < 0.001 and p = 0.001, respectively). The majority (64.2%) of the laparoscopic procedures were performed by a trainee. There was no difference in the operating times of open versus laparoscopic surgery, or of procedures performed by trainees versus staff surgeons. Laparoscopy by trainees did not have a negative impact on complication or conversion rates. CONCLUSIONS: Laparoscopy is an established approach in abdominal procedures in children, and does not hamper surgical training.

Early endostatin treatment inhibits metastatic seeding of murine colorectal cancer cells in the liver and their adhesion to endothelial cells.

Endostatin, a carboxy-terminal fragment of collagen XVIII, potently inhibits angiogenesis and tumour growth, presumably through induction of apoptosis in endothelial cells and/or inhibition of their migration. Here we have tested how the timing of recombinant human endostatin (rh-E) administration affects its antitumour activity in a liver metastasis model of mouse C26 colorectal carcinoma cells. The effects of rh-E treatment on hepatic tumour load and on early tumour cell seeding were evaluated. Recombinant human endostatin was most effective in reducing intrahepatic tumour growth when administered prior to tumour cell inoculation. Analysis of early tumour cell seeding by using [(125)I]iododeoxyuridine-labelled C26 cells or by in vivo microscopy showed that rh-E reduced tumour cell seeding in the liver sinusoids. Recombinant human endostatin did not inhibit tumour growth when administered later than 4 days after tumour injection. Pretreatment of human umbilical vein endothelial cells with rh-E in vitro reduced C26 tumour cell adhesion under flow conditions two-fold as assessed by video microscopy and multiphoton laser scanning microscopy. Our results show that rh-E, in addition to antiangiogenic effects, reduces tumour cell adhesion in the liver sinusoids during the very early phases of metastasis formation. These data point towards a previously unknown mode of action of endostatin, that is, its ability to interfere with tumour cell seeding. Such insights may be helpful in the design of trials to improve (surgical) treatment of colorectal carcinoma and liver metastases.

Impaired healing of cutaneous wounds and colonic anastomoses in mice lacking thrombin-activatable fibrinolysis inhibitor.

Plasmin and other components of the plasminogen activation system play an important role in tissue repair by regulating extracellular matrix remodeling, including fibrin degradation. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase that, after activation, can attenuate plasmin-mediated fibrin degradation by removing the C-terminal lysine residues from fibrin, which play a role in the binding and activation of plasminogen. To test the hypothesis that TAFI is an important determinant in the control of tissue repair, we investigated the effect of TAFI deficiency on the healing of cutaneous wounds and colonic anastomoses. Histological examination revealed inappropriate organization of skin wound closure in the TAFI knockout mice, including an altered pattern of epithelial migration. The time required to completely heal the cutaneous wounds was slightly delayed in TAFI-deficient mice. Healing of colonic anastomoses was also impaired, as reflected by decreased strength of the tissue at the site of the suture, and by bleeding complications in 3 of 14 animals. Together, these abnormalities resulted in increased mortality in TAFI-deficient mice after colonic anastomoses. Although our study shows that tissue repair, including re-epithelialization and scar formation, occurs in TAFI-deficient mice, TAFI appears to be important for appropriate organization of the healing process.

Enhanced antitumour efficacy by combining conventional chemotherapy with angiostatin or endostatin in a liver metastasis model.

BACKGROUND: Tumour-induced microvascular networks have become attractive targets in cancer therapy. Strategies that target both tumour cells and vasculature have not been investigated in models of early metastatic colorectal disease. The efficacy of a combination of conventional chemotherapy with a potent angiogenesis inhibitor (endostatin or angiostatin) in a murine model of early colorectal liver metastasis was studied. METHODS: Sixty-six mice were subjected to intrasplenic injection of C26 tumour cells to induce colorectal liver metastases. Control animals received phosphate-buffered saline (n = 8) or citrate buffer (n = 8). Treatment included conventional chemotherapy (n = 9), endostatin (n = 8), high-dose (n = 5) or low-dose (one-tenth of optimal dose; n = 10) angiostatin, as well as the combination of either of these drugs with chemotherapy (n > 5). Clinical appearance was scored daily using a semiquantitative scale. Liver weight, macroscopic and histological tumour involvement (hepatic replacement area; HRA) were measured upon death at day 12. RESULTS: Treated mice displayed significantly better clinical scores than controls, except for those animals treated with low-dose angiostatin with or without chemotherapy. Treatment with conventional chemotherapy resulted in a decrease in HRA from 42.3 to 29.1 per cent (P < 0.001). The addition of angiostatin or endostatin to conventional chemotherapy improved antitumoral efficacy, in a multiplicative manner, resulting in a HRA of approximately 3.5 per cent (P < 0.001). CONCLUSION: The addition of angiostatin or endostatin to conventional chemotherapy enhanced antitumoral efficacy in a murine model of early colorectal liver metastasis.

Arthrodesis after osteosynthesis and infection of the ankle joint.

Nineteen patients with a severely infected ankle joint after previous osteosynthesis were treated with arthrodesis in our institution. Their notes and X-rays were reviewed. Goals of treatment were eradication of infection by aggressive débridement of infected tissues, obtaining adequate soft-tissue coverage, preservation/restoration of bonelength, and finally consolidation of the arthrodesis. Thirteen men and six women were treated, with a median age of 46 (17-69) years. Arthrodesis took place after a median of 6 months (0.5-40) post-accident, and after one to six earlier operative procedures. Primarily there had been four bimalleolar, five trimalleolar and ten pilon tibial fractures. Fifteen fractures were open with severe soft tissue damage. Seven free muscle transfers were performed, and ten cancellous bone graftings. Finally 29 attempts at arthrodesis were performed. Ultimately we had to perform two amputations. After a mean follow up of 3.5 years, one patient has an aseptic but asymptomatic pseudarthrosis, for which no further surgery is scheduled. Sixteen extremities are free from infection while full weightbearing is possible. The limb-threatening problem of deep infection after osteosynthesis of an ankle fracture can be resolved by consistent but prolonged treatment. After successful arthrodesis a weightbearing extremity without infection remains in the majority of cases.

[Free muscle transplantation after complex fractures of the lower leg and ankle: results from 32 patients]. / Vrijespiertransplantatie na gecompliceerde fracturen van onderbeen en enkel; ervaringen bij 32 patiënten.

OBJECTIVE: To describe the results of free vascularized muscle transfer for treatment of complications of severe open fractures of the distal tibia and ankle region. DESIGN: Retrospective. METHODS: In 1994-1998, 32 patients underwent free muscle transfer for soft tissue reconstruction after high energy injury to the distal tibia and ankle region. In six patients reconstruction took place within one week after the injury (group 1); 13 patients underwent free muscle transfer in combination with (re)osteosynthesis and cancellous bone grafting for treatment of infected (defect) pseudarthrosis (group 2); chronic posttraumatic osteitis existed for an average of 12 years in six patients (group 3); in seven patients the indication was determined by unstable scar tissue, cosmetic reasons, or as preparation for corrective osteotomy (group 4). RESULTS: Three haematomas and two thrombembolic complications were encountered. Amputation could not be avoided in three patients. Consolidation of the fracture, pseudarthrosis or arthrodesis and/or eradication of infection was accomplished in all remaining 29 patients. An average of 2.2 (range: 0-7) reinterventions were additionally needed in groups 1-3, including reosteosynthesis, sequestrectomy, cancellous bone grafting and/or arthrodesis. CONCLUSION: In case of severe open fracture of the distal tibia and/or ankle region, free vascularized muscle transfer provided cover of the fracture or pseudarthrosis region, thereby improving local vascularisation. This treatment protects the underlying bone, enhances osteogenesis and improves resistance to infection.

First trimester development of human chorionic villous vascularization studied with CD34 immunohistochemistry.

Normal chorionic villous vascularization is essential for the undisturbed development of pregnancy. Defective vasculogenesis may play a role in pathological pregnancy. To assess pathological chorionic villous vascularization, normal vascularization has to be defined first. Few data are available on this topic. The aim of this study was therefore to investigate normal chorionic villous vascularization in ultrasound-dated first trimester pregnancies from week 5 menstrual age to week 12 (n = 41), using quantitative CD34 immunohistochemistry. Two important processes in chorionic villous vascularization were quantitatively illustrated: (i) maturation, reflected by an increase of the total number of luminized vessels as opposed to non-luminized haemangioblastic cords and (ii) margination, due to a decrease of villous stromal area and an increase of total villous vascular area. The percentage of villous stromal area occupied by vascular elements (area difference %) increased from 0.7% in week 5-2.5% in week 10. Therefore, the area of the villous stroma occupied by vascular elements increases and the vessels are situated closer to the trophoblastic layer suitable for fetal-maternal exchange. There was also a trend in increased number of peripheral vessels (2.0 in week 5 to 4.6 in week 10), supporting both developmental mechanisms. In conclusion, in exactly dated normal human first trimester pregnancies, development of the chorionic villous vascular system seems to be mostly characterized by maturation of luminized vessels from primitive haemangioblastic cords, and margination to a situation of peripherally located vessels.