Hugues Legendre

Refined prognostic evaluation in colon carcinoma using immunohistochemical galectin fingerprinting

Knowledge of the expression of the galectins in human colon carcinomas is mainly restricted to galectin‐3 and, to a lesser extent, galectin‐1. The current study analyzed the prognostic values contributed by galectin‐1, galectin‐3, galectin‐4, and galectin‐8 in cases of colon carcinoma.

Upregulation of galectins-1 and -3 in human colon cancer and their role in regulating cell migration

To probe the potential contribution of β‐galactoside‐contributing epitopes and receptor proteins (gal‐1 and gal‐3) to colon malignancy, we first examined the expression of galectins and binding sites in clinical specimens by lectin and immunohistochemistry. Sixty‐seven colonic surgical resections were studied, including 10 normal, 10 mild dysplasias, 10 severe dysplasias and 37 cancers. gal‐1 and gal‐3 were expressed in variable amounts in the epithelial cells and the connective tissue of normal colon. Their expression significantly increased with the degree of dysplasia, suggesting that gal‐1 and gal‐3 and their binding sites are related to malignant progression, while gal‐8 has been associated with suppressor activity. To study the functional aspects, the influence of these galectins on the migration of 4 human colorectal cancer cell lines (HCT‐15, LoVo, DLD‐1, CoLo201) was studied. In agreement with histopathologic monitoring, these tumor cells were found to produce gal‐3, while only CoLo201 was positive for gal‐1. Except for DLD‐1 and gal‐1, the lines exhibited gal‐1 binding sites on the surface, prompting study by computer‐assisted videomicroscopy of the effect on cell migration of the presence of galectin on the culture substrate. The level of cell migration for HCT‐15, LoVo and CoLo201 cells was significantly reduced by 0.15 μg/cm2 gal‐1, and the presence of a blocking antibody at least reduced this effect. gal‐3 significantly reduced cell migration in all 4 of the in vitro cell lines.

Galectin-8 expression decreases in cancer compared with normal and dysplastic human colon tissue and acts significantly on human colon cancer cell migration as a suppressor

Background and aims: Galectins are β-galactoside binding proteins. This ability may have a bearing on cell adhesion and migration/proliferation in human colon cancer cells. In addition to galectins-1 and -3 studied to date, other members of this family not investigated in detail may contribute to modulation of tumour cell features. This evident gap has prompted us to extend galectin analysis beyond the two prototypes. The present study deals with the quantitative determination of immunohistochemical expression of galectin-8 in normal, benign, and malignant human colon tissue samples and in four human colon cancer models (HCT-15, LoVo, CoLo201, and DLD-1) maintained both in vitro as permanent cell lines and in vivo as nude mice xenografts. The role of galectin-8 (and its neutralising antibody) in cell migration was investigated in HCT-15, LoVo, CoLo201, and DLD-1 cell lines. Methods: Immunohistochemical expression of galectin-8 and its overall ability to bind to sugar ligands (revealed glycohistochemically by means of biotinylated histochemically inert carrier bovine serum albumin with α- and β-d-galactose, α-d-glucose, and lactose derivatives as ligands) were quantitatively determined using computer assisted microscopy. The presence of galectin-8 mRNA in the four human colon cancer cell lines was examined by reverse transcriptase-polymerase chain reaction. In vitro, cellular localisation of exogenously added galectin-8 in the culture media of these colon cancer cells was visualised by fluorescence microscopy. In vitro galectin-8 mediated effects (and the influence of its neutralising antibody) on migration levels of living HCT-15, LoVo, CoLo201, and DLD-1 cells were quantitatively determined by computer assisted phase contrast microscopy. Results: A marked decrease in immunohistochemical expression of galectin-8 occurred with malignancy development in human colon tissue. Malignant colon tissue exhibited a significantly lower galectin-8 level than normal or benign tissue colon cancers; those with extensive invasion capacities (T3–4/N+/M+) harboured significantly less galectin-8 than colon cancers with localised invasion capacities (T1–2/N0/M0). The four experimental models (HCT-15, LoVo, CoLo201, and DLD-1) had more intense galectin-8 dependent staining in vitro than in vivo. Grafting the four experimental human colon cancer models onto nude mice enabled us to show that the immunohistochemical expression of galectin-8 was inversely related to tumour growth rate. In vitro, galectin-8 reduced the migration rate of only those human experimental models (HCT-15 and CoLo201) that exhibited the lowest growth rate in vivo. Conclusions: Expression of galectin-8 correlated with malignancy development, with suppressor activity, as shown by analysis of clinical samples and xenografts. In vitro, only the two models with low growth rates were sensitive to the inhibitory potential of this galectin. Future investigations in this field should involve fingerprinting of these newly detected galectins, transcending the common focus on galectins-1 and -3.

Prognostic Values of Galectin-3 and the Macrophage Migration Inhibitory Factor (MIF) in Human Colorectal Cancers

This study aims to investigate whether the immunohistochemical levels of expression of galectin-3 and the macrophage migration inhibitory factor (MIF) are associated with prognostic values in human colorectal tumors. This was performed on 99 specimens including 69 colorectal tumors (17 Dukes A, 19 Dukes B, 15 Dukes C and 18 metastatic tumors that we labeled as D), 10 hepatic metastases from colorectal cancers and 20 normal specimens (biopsies). The immunohistochemical levels of expression of MIF and galectin-3 were quantified on routine histological slides by means of computer-assisted microscopy. Separate analyses were performed on epithelial and connective tissue. The levels of expression of both MIF and galectin-3 were very significantly higher in epithelial tumor tissue when compared with normal epithelial specimens. A positive and significant correlation between MIF and galectin-3 expression was evidenced in connective tumor tissue, and in particular in the cases associated with short survival periods (less than 5 years). In the case of the Dukes A or B tumors, we established two new prognostic groups (labeled I and II) on the basis of the levels of galectin-3 expression measured in the tumor epithelium. In the case of the Dukes C or D tumors, we established two other prognostic groups (labeled III and IV) on the basis of the levels of MIF expression measured in the connective tissue. Kaplan-Meyer analyses confirmed the additional prognostic values (as compared with conventional clinical staging) given by this new classification (groups I to IV). They show that the Dukes A or B tumors characterized by low levels of galectin-3 expression in the tumor epithelium are associated with significantly better prognoses than those characterized by high levels. In addition, the Dukes C or D tumors characterized by high levels of MIF expression in the connective tumor tissue are associated with significantly better prognoses than those characterized by low levels. In conclusions, MIF and galectin-3 expression levels in colorectal tumors are related to their levels of biological aggressiveness. These markers could be used to identify patients at risk, for whom more aggressive adjuvant therapy seems to be indicated.

Binding Sites for Lewis Antigens Are Expressed by Human Colon Cancer Cells and Negatively Affect Their Migration

In colon cancer, endothelial cell selectins can promote tumor cell attachment via interactions with sialylated Lewis antigens present at the surface of tumor cells, thereby facilitating tumor cell arrest and transmigration into the extravascular space. However, it is not known whether Lewis antigens interact with colon tumor cells and modify their migration. Our aim was to detect the presence of binding sites on human tumor cells for Lewisa/x antigens and their sialylated derivatives in vitro and in vivo and to analyze their influence on migration of colon cancer cells. The immunocytochemical and histochemical levels of expression of the four Lewis antigens were quantitatively determined in four human colon cancer cell lines and in in vivo nude mice xenografts. The levels of expression of specific binding sites for these sugar epitopes were determined by synthetic neoglycoconjugates. The influence of binding of these carbohydrate ligands on cancer cell migration was quantitatively evaluated by computer-assisted phase-contrast videomicroscopy performed on Matrigel culture supports either left uncoated or coated with neoglycoconjugate presenting synthetic Lewisa, sialyl Lewisa, Lewisx, or sialyl Lewisx antigens. The influence of the calcium concentration in the culture medium on the Lewis antigen–mediated effects was checked. Human colon cancer cells expressed significant amounts of specific binding sites detected by the synthetic probes in addition to the oligosaccharide epitopes. The expression levels differed considerably between the four cell lines and between in vitro and in vivo specimens. Cell migration analysis revealed that the four Lewis antigens markedly decreased the levels of migration of the HCT-15 and LoVo cancer cells. This effect depends on the calcium concentration in the culture medium. Binding sites for Lewis epitopes are present on colon cancer cells. The functional relevance of these sites is indicated by the negative influence on cell migration of a matrix containing the oligosaccharides as ligand parts.

Long-term survival of patients downstaged by oxaliplatin and 5-fluorouracil combination followed by rescue surgery for unresectable colorectal liver metastases.

OBJECTIVES To evaluate long-term survival of patients resected for primarily unresectable colorectal liver metastases downstaged by systemic chemotherapy. METHODS Among a group of 82 patients with advanced colorectal cancer, 39 had unresectable liver metastases. After treatment with systemic 3-weekly 5FU/folinic acid/oxaliplatin chemotherapy, the outcome of 11 patients made resectable thanks to chemotherapy was compared to that of 28 patients who were not. Criteria for non-resectability consisted of diffuse bilobar invasion with inability to achieve complete resection, unilobar or bilobar invasion plus vascular extension (invasion of inferior vena cava or 2 supra-hepatic veins plus continuity with the 3rd) or involvment of hepatic pedicle. Before and after surgery, CT scan evaluation was performed every 2 months. Progression free survival was defined as the time between starting chemotherapy and recurrence of the disease. We used Kaplan-Meier survival curves and log-rank test for comparisons, P values were two-sided and considered significant if<0.05. RESULTS Progression free survival times were 14 and 6 months, median overall survival were 60 and 18.5 months, respectively, in favour of secondary resected subjects. CONCLUSION Considering the magnitude of the survival benefit, one may question the need and feasibility for trials to assess more formally the impact of surgery in that setting.

Venous thrombosis associated with catheter-related mediastinal perforation due to catheter mispositioning.

A 57-year old woman with an epidermoid carcinoma of the rhinopharynx was referred at our institution for treatment. A Totally Implantable Venous Access Device (BRAUN, Model CELSITE ST301) was placed percutaneously in the left subclavian vein under radioscopy. The postoperative chest x-ray showed no complications but the tip of the catheter was situated too high. Its functionality was satisfactory and we decided to keep it in place and to use it for chemotherapy administration. Twenty months after her last treatment, she was admitted for asthenia and pyrexia. She complained about pain in her left superior arm during TIVAD testing. A cavography was performed and showed an extravasation of the contrast solution in the anterior mediastinum (Fig. 1). A thoracic CT was then performed to confirm the diagnosis and to exclude mediastinitis. It showed a complete thrombosis of the left brachio-cephalic trunk but no sign of mediastinitis (Fig. 2). TIVAD was removed and the patient was treated with low molecular weight heparin. The patient was monitored in the intensive unit care for 24 hours. The clinical evolution was uneventful and the patient was discharged on the second day. She presents without any complications at 21 months follow-up.