Stereotactic body radiation therapy for liver metastases in oligometastatic disease.

Oligometastatic cancers designate cancers in which the number of metastases is less than five, corresponding to a particular biological entity whose prognosis is situated between a localized and metastatic disease. The liver is one of the main sites of metastases. When patients are not suitable for surgery, stereotactic body radiotherapy provides high local control rate, although these data come mainly from retrospective studies, with no phase III study results. The need for a high therapeutic dose (biologically effective dose greater than 100Gy) while respecting the constraints on the organs at risk, and the management of respiratory movements require expertise and sufficient technical prerequisites. The emergence of new techniques such as MRI-guided radiotherapy could further increase the effectiveness of stereotactic radiotherapy of liver metastases, and thus improve the prognosis of these oligometastatic cancers.

Radiation therapy of the primary tumour and/or metastases of digestive metastatic cancers.

Metastatic gastrointestinal cancer is not an uncommon situation, especially for pancreatic, gastric, and colorectal cancers. In this setting, few data are available on the impact of the treatment of the primary tumour. Oligometastatic disease is associated with longer survival in comparison with more advanced disease. Metastasis-directed therapy, such as stereotactic body radiotherapy, seems related to better outcomes, but the level of evidence is low. In most tumour locations, prospective data are very scarce and inclusion in ongoing trials is strongly recommended.

[Influence of epigenetic in Sjögren's syndrome]. / Épigénome et syndrome de Gougerot-Sjögren.

Sjögren's syndrome (SS) is a systemic autoimmune epithelitis with a major female incidence, and characterized by a dry syndrome, impaired quality of life, visceral involvement, and lymphoma for the most aggressive cases. During this process, epithelial cells acquire the capacity to produce cytokines, chemokines, and autoantigens which can in turn be presented to the immune system. Consequently, this epithelitis is accompanied by lymphocytic infiltrations leading to the formation of pseudo-follicles in which self-reactive B lymphocytes are present. The recent integration of genomic and especially of epigenomic data, which make it possible to analyze the different cellular partners, opens new perspectives and allows to a better understanding of this complex and still incurable disease.

Epigenetic modifications in salivary glands from patients with Sjögren's syndrome affect cytokeratin 19 expression.

Sjögren's syndrome (SS) is a chronic autoimmune epithelitis, and several lines of experiments indicate that multifactorial factors contribute to salivary gland epithelial cells (SGEC) dysfunctions including a combination of environmental factors, lymphocytic infiltrations, genetic predispositions as well as epigenetic defects. Such statement is reinforced by the observation that global DNA methylation (5MeCyt) is altered in minor salivary glands from pSS patients and that such defect is associated cytokeratin 19 (KRT19) overexpression. An epigenetic deregulation of the KRT19 gene was further tested by treating the human salivary gland (HSG) cell line with the DNA demethylating agent 5-azacytidin, and with the histone acetylase inhibitor trichostatin A. Blocking DNA methylation, but not histone acetylation, with 5-azacytidin was associated with KRT19 overexpression at both transcriptional and protein level. Next, analysis of the CpG genome-wide methylome array in the KTR19 locus from long term cultured SGEC obtained from 8 pSS patients revealed a more reduced DNA methylation level in those patients with defective global DNA methylation. Altogether, our data, therefore, suggest that alteration of DNA methylation in SGEC may contribute to pSS pathophysiology in part by controlling the expression of KRT19.

[Classification of urinary calculi in a pediatric population from Brittany]. / Proposition de "profils types" des calculs urinaires dans une population pédiatrique bretonne.

INTRODUCTION: Urinary lithiasis in children is relatively seldom in France as in industrialized countries. The determination of their etiology based on their composition may lead to a better treatment. METHOD: One hundred and eight urinary calculi from 6 months through 18-year-old children were analyzed by using spectrophotometry, in order to specify their structure. Six groups were evidenced through a multidimensional analysis based on the presence of components weighing at least 5% of the total. RESULTS: The youngest children affected were mostly boys, and the sex ratio switched after 12.5 years. Above 14 years of age, the number of calculi significantly raised. Their composition varied with the gender, and their localization with the age. Finally a correlation between infection and composition of the calculus was shown in our study. CONCLUSION: The classification of calculi among six groups according to their composition, along with clinical informations and morphologic studies, has proven its value in determining the etiology of the lithiasis. These data help to better understand the kind of lithiasis that may be observed and the physiopathology of the mechanism explaining it from the gender and age.

Prognosis factors of survival time in patients with glioblastoma multiforme: a multivariate analysis of 340 patients.

BACKGROUND: The prognosis of glioblastoma multiforme remains poor despite recent therapeutic advances. Several clinical and therapeutic factors as well as tumour characteristics have been reported as significant to survival. A more efficient determination of the prognostic factors is required to optimize individual therapeutic management. The aim of our study was to evaluate by univariate then multivariate analysis the factors that influence prognosis and particularly survival. METHODS: Data of 340 patients with newly-diagnosed GBM were retrospectively analyzed. Univariate analysis of prognosis factors of survival time was performed. Factors that seemed determinant were evaluated by Kaplan-Meier survival curves. Finally, the significant factors found in univariate analysis were tested in multivariate analysis using the COX regression method. FINDINGS: Using multivariate analysis, the following factors were found to influence survival: radiotherapy was the predominant factor followed by radical surgery, tumour location, age and chemotherapy. Patients treated with temozolomide had a markedly better survival rate than patients treated with other chemotherapies (Log-rank test P < 0.005). The values of GBM type (de novo or secondary), as well as repeated surgery and partial surgery (vs. simple biopsy) were suggested by univariate analysis but not confirmed by the COX regression method. After radical surgery, progression-free survival was correlated to overall survival (r = 0.87, P < 10e-5). CONCLUSIONS; The influence of radiotherapy on survival was greater than the influence of age, an argument supporting the proposition of radiotherapy for patients until at least age 70. In the case of recurrence, the correlation between overall survival and progression-free survival is an important factor when considering the therapeutic options. Initial radical surgery and repeated procedures dramatically influence survival. The benefit of partial surgery remains difficult to evaluate. Partial surgery could be used to decrease intracranial pressure and to minimize residual tumours in order to enable treatment by chemotherapy and radiotherapy. The value of temozolomide treatment was confirmed.

B cell-ablative therapy: where are we now?

Based on their multifaceted functions, B cells participate in several pathological settings such as lymphoproliferative disorders, autoimmune diseases and graft rejection. B cell-ablative therapy has thus emerged as a mainstay in these diseases. A number of anti-B cell antibodies (Abs) have been generated, among which anti-CD20 Abs appear to be efficient. Rituximab (RTX) is one of these anti-CD20 monoclonal Abs. Originally approved for the treatment of non-Hodgkin lymphoma, RTX is now being administered in other malignant proliferations, applied to an increasing number of autoimmune diseases and required to prevent rejection of a graft. Although this medication is remarkably safe, a handful of laboratory tests have been proposed to monitor RTX-treated patients. The efficacy in different diseases, and the emergence of new anti-CD20 Abs raise many questions. Thus, their detailed understanding can lead to a better issue for inhibition of immune responses.

Recombinant humanised anti-HER2/neu antibody (Herceptin) induces cellular death of glioblastomas.

Glioblastoma multiforme (GBM) remains the most devastating primary tumour in neuro-oncology. Targeting of the human epithelial receptor type 2 (HER2)-neu receptor by specific antibodies is a recent well-established therapy for breast tumours. Human epithelial receptor type 2/neu is a transmembrane tyrosine/kinase receptor that appears to be important for the regulation of cancer growth. Human epithelial receptor type 2/neu is not expressed in the adult central nervous system, but its expression increases with the degree of astrocytoma anaplasia. The specificity of HER2/neu for tumoral astrocytomas leads us to study in vitro treatment of GBM with anti-HER2/neu antibody. We used human GBM cell lines expressing HER2/neu (A172 express HER2/neu more than U251MG) or not (U87MG) and monoclonal humanised antibody against HER2/neu (Herceptin). Human epithelial receptor type 2/neu expression was measured by immunohistochemistry and flow cytometry. Direct antibody effect, complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity were evaluated by different cytometric assays. We have shown, for the first time, the ability of anti-HER2/neu antibodies to induce apoptosis and cellular-dependent cytotoxicity of HER2/neu-expressing GBM cell lines. The results decreased from A172 to U251 and were negative for U87MG, in accordance with the decreasing density of HER2/neu receptors.

Comparison of cell-ELISA, flow cytometry and Western blotting for the detection of antiendothelial cell antibodies.

OBJECTIVE: There is still great uncertainty in the detection of antiendothelial cell antibodies (AECA). The aim of our study was to compare the results obtained using different methods. METHODS: Sera were obtained from 71 patients with a variety of vasculitides. Three assay methods were used: cell ELISA, flow cytometry (FACS) and Western blot (WB). RESULTS: In the ELISA 12/17 patients with systemic lupus erythematosus (SLE), 1/12 with Churg Strauss (CS) disease, 3/12 with micropolyarteritis (MPA) and 5/30 with Wegener's granulomatosis (WG) tested positive. Most of the sera that were positive on ELISA were not by FACS. Among the negative sera, 50% of WG, 40% of MPA, 20% of CS and 40% of SLE became positive on WB. There were some specific patterns of reactivity for a given disease, so that some bands could be assigned to a disease. CONCLUSION: The discrepancies in the results may most probably be accounted for by differences between the antigenic preparations. Caution must thus be exercised when interpreting the results of any of these three tests.

Functional heterogeneity of anti-endothelial cell antibodies.

While it has been claimed that some anti-endothelial cell antibodies (AECA) activate EC, there is also evidence that others trigger apoptosis. To address the issue of whether activation is a prerequisite for AECA-mediated apoptosis of EC, 23 AECA-positive sera were evaluated for their ability to induce activation and/or apoptosis. Activation was defined as an over-expression of E-selectin and intercellular adhesion molecule 1. Optical microscopy, annexin V binding, hypoploid cell enumeration, and determination of poly (ADP-ribose) polymerase cleavage-related products were used to assess apoptosis. Four functional profiles were defined: 10 sera promoted activation and apoptosis (act+/apo+), one was act+/apo-, six act-/apo+, and the remaining six act-/apo-. The reduced membrane expression of thrombomodulin was associated with apoptosis, rather than activation. Caspase-3 was implicated in the two models of apoptosis, the ratios of several survival proteins to Bax decreased, regardless of the ability of apo+ AECA to activate the cells, while radical oxygen species did not appear to be involved. Furthermore, it occurred that macrophages engulfed EC treated with apoptosis-promoting AECA, but not those incubated with AECA that did not induce apoptosis. Hence, AECA represent an extremely heterogeneous family of autoantibodies, not only because of the variety of their target antigens, but also the subsequent diversity of their effects.

Anti-endothelial cell reactivity, the unresolved enigma.

Not only are some anti-endothelial cell antibodies (AECA) directed to thus far unidentified cell membrane structures, but some ot them recognize "planted" antigens and possibly ligand-receptor complexes. The functional heterogeneity of AECA is widely acknowledged: part of them activate the complement, mediate antibody-dependent cell cytotoxicity of trigger the production of tissue procoagulant factor. It has also recently been established that a proportion of AECA have the capacity to induce apoptosis of their target cells. In fact, the most direct demonstration of the pathogenicity of AECA is the autoantibody-induced murine model of vasculitis.

False positivity in a cyto-ELISA for anti-endothelial cell antibodies caused by heterophile antibodies to bovine serum proteins.

BACKGROUND: ELISAs with fixed endothelial cells or cell lines are widely used screening tests for anti-endothelial cell antibodies (AECAs), but spurious increases occur. We examined interferences by heteroantibodies and means to eliminate them. METHODS: AECAs were measured by ELISA on fixed layers of the human endothelial cell line, EA.hy 926, in a panel of 60 patient serum samples diluted in bovine serum albumin. Heteroantibodies against fetal calf serum (FCS) proteins were demonstrated and characterized in an ELISA-the interference assay-that used FCS-coated plates and Tween 20-containing buffer as blocking agent and sample diluent, as well as by immunoblotting. RESULTS: In 12 of 60 patient serum samples, spurious increases of AECA titers were produced by endogenous antibodies reacting with FCS proteins from culture medium that were coated onto the solid-phase at the time of cell plating. This mechanism of interference was supported experimentally by exposing extracellular matrix, varying cell density, and incubating wells with FCS alone. The heterophile antibodies were mainly IgG and IgA, and in inhibition experiments, they recognized serum proteins from goat, sheep, and horse. Washing cells free of FCS before plating, or adding FCS (100 mL/L) to the patient sample diluent eliminated spurious signals from all 30 tested sera, but the latter method had practical advantages. CONCLUSIONS: Antibodies against animal serum proteins are a frequent cause of erroneous results in cyto-ELISAs. The interference can be eliminated by simple antibody absorption in FCS-containing dilution buffer.

Anti-endothelial cell antibody binding makes negatively charged phospholipids accessible to antiphospholipid antibodies.

OBJECTIVE: Anti-endothelial cell autoantibodies (AECA) are often associated with antibodies to anionic phospholipids (PL), such as phosphatidylserine (PS). Yet, beta2-glycoprotein I (beta2GPI)-dependent anti-PL antibodies (aPL) do not have access to their target antigens on the membrane of endothelial cells (EC). Given that AECA are capable of exposing PS and, thereby, initiating apoptosis, we explored the relationships between AECA, beta2GPI, and aPL on the surface of EC. METHODS: Human EC were incubated with mouse AECA monoclonal antibodies, and the translocation of PS was established through the binding of annexin V, which binds specifically to PS. A rabbit anti-beta2GPI antibody and biotin-conjugated F(ab')2 aPL derived from 3 patients were also used to detect beta2GPI on the cells. RESULTS: Twenty percent to 36% of the cells expressed anionic PL following incubation with AECA, as revealed by the binding of annexin V and beta2GPI. The proportion of anionic PL-expressing EC (up to 90%) correlated with the period of incubation of EC with AECA and depended on the dose of AECA. Bound aPL resided exclusively within the AECA-positive EC population. CONCLUSION: Based on our findings, AECA may be pathogenic. Some of them may even have the potential to induce production of aPL.

The binding of some human antiendothelial cell antibodies induces endothelial cell apoptosis.

The pathogenic role of antiendothelial cell antibodies (AECA) remains unclear. They are frequently associated with antibodies to anionic phospholipids (PL), such as phosphatidylserine (PS), which is difficult to reconcile with the distribution of PL molecular species within the plasma membrane. Since it is already known that PS is transferred to the outer face of the membrane as a preclude to apoptosis, the possibility exists that apoptosis is initiated by AECA. AECA-positive/anti-PL antibody-negative sera from eight patients with systemic sclerosis (SS) and 21 control patients were evaluated. Endothelial cells (EC) were incubated with AECA and the exposure of PS was established through the binding of annexin V. Hypoploid cell enumeration, DNA fragmentation, and optical and ultrastructural analyses of EC were used to confirm apoptosis. Incubation of EC with AECA derived from six of eight patients with SS led to the expression of PS on the surface of the cells. This phenomenon was significantly more frequent in SS (P < 0.04) than in control diseases. The redistribution of plasma membrane PS preceded other events associated with apoptosis: hypoploidy, DNA fragmentation, and morphology characteristic for apoptosis. Apoptosis-inducing AECA did not recognize the Fas receptor. We conclude that AECA may be pathogenic by inducing apoptosis.

Two populations of endothelial cell antibodies cross-react with heparin.

As endothelial cells (EC) express heparin-like glycosaminoglycans, such as heparan sulfate, it was essential to investigate the relation of anti-EC antibody (AECA) to heparin reactivity. AECA were detected in 43 of 131 autoimmune sera and anti-heparin antibodies (AHA) in 25. These autoimmune reactivities were significantly associated (P corrected < 0.0005). Seven AECA-positive/AHA-positive and three AECA-negative/AHA-positive sera were affinity-purified using protein G column followed by a heparin-Sepharose column. Two populations of AECA were recovered from the second column. One was eluted with 0.4 M NaCl which bound to EC and to solid-phase heparin with low affinity, but not to soluble heparin. The second population of AECA, which was eluted with 4 M guanidine HCl/2 M NaCl, recognized EC and solid-phase heparin with high affinity, but also soluble heparin. The latter population of AECA might thus be an important cause of autoimmune vascular thrombosis in systemic diseases.

Anti-endothelial cell antibody, thrombomodulin, and von Willebrand factor in idiopathic inflammatory myopathies.

Sera from 19 patients with idiopathic inflammatory myopathy (IIM) were examined for the presence of anti-endothelial cell antibodies (AECA) by an immunoglobulin G-specific cellular enzyme-linked immunosorbent assay. The mean binding index of AECA was found to be 37.7% +/- 26.5% for the patients, compared with a mean of 7.2% +/- 2.7% for normal controls (P < 0.04). Levels of thrombomodulin, von Willebrand factor antigen, and serum creatine kinase were also shown to be augmented. Interestingly, positive correlations between AECA on the one hand and Raynaud's phenomenon and interstitial lung disease on the other were demonstrated. Given that the pathogenesis of IIM remains uncertain, these findings may be of importance.