Biologic therapies for plaque type psoriasis in patients with previous malignant cancer: long-term safety in a single- center real-life population.

INTRODUCTION: This is an Italian single-center retrospective study evaluating safety and efficacy of biologic agents in psoriatic patients with a previous diagnosis of malignant cancer. AIM: Management of moderate and severe psoriasis patients with a past medical history of malignancies could be difficult because biologic agents are historically associated with a presumptive increased risk of neoplastic reactivation or of a new incoming cancer. The aim of this study is to assess the safety of biologics in patients with a previous cancer diagnosis. MATERIAL AND METHODS: The study analyzed 16 moderate to severe psoriasis patients with a diagnosis of malignant cancer in the previous 10 years treated with biologic agents for up to at least 96 weeks. In five of these patients, cancer was diagnosed in the previous 5 years. RESULTS: We observed a rapid decrease in PASI (psoriasis area severity index) reaching a 90% improvement in 100% of patients. Oncologic follow up did not show any worsening or reactivation of cancer during the entire observation period. No new malignancies were observed in the analyzed sample. CONCLUSIONS: Biologic agents in our experience have demonstrated to be safe and effective in psoriatic patients with a past medical history of malignant cancer.

Growth of ovarian-carcinoma cell lines at physiological folate concentration: effect on folate-binding protein expression in vitro and in vivo.

Ovarian-carcinoma cell lines (OVCAR3, IGROVI, OVCA432, SW626 and SKOV3), grown in standard medium containing supra-physiological (2.3 microM) folate concentration, display different levels of reactivity with the anti-folate-binding-protein (FBP) monoclonal antibody MOv18, which recognizes the alpha-isoform of the protein. Gel-filtration and absorption experiments indicated that on IGROVI cells this molecule accounts for all folic-acid binding at nanomolar concentrations. The aim of the study was to investigate the effect of extracellular folate levels on cells adapted to grow in medium containing physiological folate concentration (20 nM). By the ternary complex assay, all cell lines showed a marked depletion of intracellular reduced folates, compared with those in standard folate medium. The monitoring of FBP by MOv18 showed on IGROVI cells a transient up-regulation of the protein, whereas on the other cell lines, except SKOV3, no changes were detected. These data suggest that in these cells further over-expression of the molecule cannot generally be induced by lowering the extracellular folate concentration. On SKOV3, Scatchard analysis of 125I-MOv18 binding, as well as the evaluation of total folate binding capacity, showed a 2- to 3-fold stable increase of FBP expression after long-term growth in low-folate medium. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated in these cells a 1.5-fold increase in alpha-FBP mRNA. SKOV3 cells, maintained in vitro in medium containing supraphysiological and physiological (i.e., low-folate) concentrations were injected into nude mice. Weight differences, though not statistically significant, were observed in favour of low-folate-derived tumors. Immunohistochemical and immunochemical analysis of the tumor samples showed that in SKOV3 cells the receptor modulation can also be induced by restoring the physiological folate concentration in vivo.

Polarization of the alpha 6 beta 4 integrin in ovarian carcinomas.

By immunizing a BALB/c mouse with a human ovary-carcinoma cell line (IGROV1), grown intraperitoneally in nude mice, a monoclonal antibody (MAb), designated MAR6, was produced and characterized. Immunofluorescence on the immunizing cell line showed a specific labelling by MAR6 at the cell-to-cell contact points. In addition, MAR6 was found to immunoprecipitate the alpha 6 beta 4 integrin complex. Competition tests with MAbs anti-alpha 6, anti-beta 4, anti-beta 1 sub-units demonstrated that the recognized sub-unit is alpha 6. Indirect immunofluorescence on various cell lines gave MAR6 as positive only on alpha 6-positive lines (IGROV1, OVCAR3, SW626, SKOV3, ME4405, Calu3, N592, MDA468, A431 and HT29). Moreover, on IGROV1 and OVCAR3 ovary-carcinoma cells, which normally grow either adhering to the culture flask or forming clumps in suspension in the medium, MAR6 selectively stained the connection points between the cells in clumps, where, in the same position, the presence of the beta 4 sub-unit, laminin and fibronectin was detected. On the contrary, the beta 1 sub-unit was distributed over the whole cell membrane. The same pattern of labelling by these MAbs was observed in 2 cases of ovarian-carcinoma cells present in ascitic fluids obtained from patients. Immunoperoxidase tests performed on cryosections of various normal tissues showed specific reactivity of MAR6 on basal or basolateral membranes of epithelial cells. On cryosections of ovarian tumors, MAR6 reactivity correlated with the degree of tumor differentiation. Indeed, in benign and well-differentiated tumors, a strong basal or basolateral labelling only of cells surrounding the neoplastic nodules was found. On the contrary, on undifferentiated tumors the inner part of the tumor nodules was also progressively labelled, whereas the staining on the border was weak and discontinuous as a result of the alpha 6 sub-unit dispersion on the tumor cell surface.

Isolation and biochemical characterization of the soluble and membrane forms of folate binding protein expressed in the ovarian carcinoma cell line IGROV1.

The human ovarian carcinoma cell line, IGROV1, produces two forms of folate binding protein (FBP), the membrane form that is anchored to the cell surface by a glycosylphosphatidylinositol tail and the soluble form that is shed into the tissue culture medium. Both forms are recognized by the monoclonal antibodies MOv18 and MOv19. Here we describe their purification and biochemical characterization. The purified soluble protein appeared as a single band with an apparent Mr of 36 kDa after SDS-PAGE, whereas the membrane form appeared as a single band with an apparent Mr of 38 kDa. The size difference between the two forms of FBP was confirmed by gel filtration of both the native and the N-glycanase-treated proteins. Both purified proteins had equal capacity to bind folic acid. The immunological cross-reactivity and the folic acid binding capability of the FBPs extracted from IGROV1 gave more evidence of the possible existence of a precursor-product relationship between them.

Metabolic effects of benzoate and sorbate in the yeast Saccharomyces cerevisiae at neutral pH.

Preincubation of yeast cells in the presence of benzoate or sorbate at an extracellular pH value of 6.8 elicited a set of metabolic effects on sugar metabolism, which became apparent after the subsequent glucose addition. They can be summarized as follows: a) reduced glucose consumption; b) inhibition of glucose- and fructose-phosphorylating activities; c) suppression of glucose-triggered peak of hexoses monophosphates; d) substantial reduction of glucose-triggered peak of fructose 2,6-bisphosphate; e) block of catabolite inactivation of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxy-kinase, but not of cytoplasmic malate dehydrogenase. On the whole this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenetic to a glycolytic state. Our data also show that, unlike former assumptions, intracellular acidification is not likely to mediate the bulk of metabolic effects of benzoate and sorbate, since under our working conditions intracellular pH kept close to neutrality.

Membrane association and shedding of the GPI-anchored Ca-MOv18 antigen in human ovary carcinoma cells.

The antigen recognized by the MOv18 MAb (Ca-MOv18) was recently shown to be a glycosylphosphatidylinositol (GPI)-linked protein. In this report we show that GPI-anchorage is not limited to IGROVI cells nor to other ovary carcinoma cell lines, but Ca-MOv18 was also found to be sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) treatment on fresh ovarian cancer cells. Furthermore, we found a heterogeneous sensitivity of Ca-MOv18 to PI-PLC cleavage, not only among the different cells studied but also in different experiments performed on the same cell line, during extended periods of time in culture. Sensitivity to PI-PLC cleavage was determined by immunofluorescence on live cells and by double-determinant radioimmunoassay of the antigen released in the supernatant. The specificity of the PI-PLC cleavage was demonstrated as follows: (a) TX114 solubilized Ca-MOv18 shifts from the detergent to the aqueous phase after treatment with PI-PLC; (b) on membrane preparations, PI-PLC specifically released a fraction of the antigen, which is distinct from the weakly associated form released by high-salt treatment; (c) Ca-MOv18 from IGROVI expressed the cross-reacting determinant (CRD), which is characteristic of GPI-linked molecules. The absence of CRD expression on the spontaneously released protein and the possibility of artificially inducing antigen shedding during the resynthesis of Ca-MOv18 which follows bacterial PI-PLC treatment are interesting points which need to be further investigated in order to understand the physiology of the Ca-MOv18 tumor antigen.

Glycolipids carrying Le(y) are preferentially expressed on small-cell lung cancer cells as detected by the monoclonal antibody MLuC1.

The monoclonal antibody MLuC1, which reacts strongly with a high percentage of small-cell lung cancers (SCLC), as well as with various human carcinomas, has been used to immunochemically characterize the recognized epitope (CaMLuC1). To this aim 3 different approaches were adopted: (1) immunoblotting/immunostaining of extracts from various tumor-cell lines; (2) inhibition of binding by purified oligosaccharides; (3) direct binding to oligosaccharide-protein conjugates. All of these experiments indicate that CaMLuC1 is present on the Le(y) blood-group structure heterogeneously expressed on various glycoproteins and glycolipids. The expression of the glycoconjugates carrying Le(y) was then analyzed on breast and lung cancers and on their normal counterparts. Our overall results suggest that SCLC produce Le(y)-active glycolipids in higher amounts compared to other tumors of the same or of a different oncotype, as well as normal lung cells, thus indicating an SCLC-specific modification of the glycosylation pathways.

Study of the biological and prognostic significance of the antigen CaMBr8 on breast carcinoma.

We previously reported that the expression on the primary tumour of the antigen CaMBr8 was related to a short survival, attributable either to higher tumour aggressiveness or a poor response to oophorectomy. To further verify the CaMBr8 prognostic value, we analysed retrospectively 862 breast cancer patients with a 19 year follow-up. In this series, CaMBr8 expression was found to be associated to some negative prognostic factors (premenopausal status, lymphnode invasion, a high number of mitosis and HER-2/neu oncoprotein expression), but had no influence on the patients' survival. Direct association with a poor prognosis was only evident in patients with lobular or mixed breast carcinoma, which however represent only a small fraction of the total breast cancers. Another possibility was that CaMBr8 could identify a subgroup of patients which did not respond to hormone therapy. To verify this hypothesis we evaluated on a second series of 116 patients the relationship between CaMBr8 expression and hormone-receptor levels. A negative association emerged which was also observed in vitro in the human breast cancer line MCF-7 treated with Sodium Butyrate, a differentiation inducer, which reduced hormone-receptor levels and increased CaMBr8 expression. In conclusion, the longer survival of CaMBr8 negative tumour patients observed in the initial study, was probably related to a better response to oophorectomy, due to the hormone-receptor level of their tumours.

Occurrence of two phosphorylated forms of yeast fructose-1,6-bisphosphatase with different isoelectric points.

Yeast fructose-1,6-bisphosphatase (EC 3.1.3.11) immunoprecipitated from glucose-derepressed wild-type cells and subjected to isoelectric focusing, appears as a unique peak, essentially homogeneous and devoid of incorporated phosphate. However, after cell incubation with glucose, two phosphorylated forms are detectable. The isoelectric point of one is higher and of the other is lower than that of the native form. In contrast, in the mutant ABYS1 which is deficient in several vacuolar proteinases (Achstetter, T., Emter, O., Ehmann, C. and Wolf, D.H. (1984) J. Biol. Chem. 259, 13334-13343), only the more acidic phospho form appears after cell incubation with glucose. However, sequence data rule out the possibility that limited proteolysis is the event responsible for the appearance of the more basic form of the phosphoenzyme. Nevertheless, time courses of glucose-induced inactivation of fructose-1,6-bisphosphatase show that the enzyme undergoes a substantially slower inactivation in the ABYS1 mutant as compared to the wild-type. These findings point to a degradative mechanism involving, besides the well-known phosphorylation, an additional as yet unknown modification which probably sensitizes the enzyme to proteolytic attack; furthermore, the enzyme responsible for such a modification seems to require one or more of the vacuolar proteinases missing in the mutant for its maturation.