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Fibronectins (FNs), polymorphic high molecular mass glycoproteins of the extracellular matrix, are known to play a major part in various processes including wound healing and oncogenic transformation. A B+FN isoform, characterised by peculiar sequence and splicing patterns,1 has been found to be widely expressed in fetal and neoplastic tissues, in sharp contrast with the highly restricted distribution in normal adult tissues, including human brain. This isoform, named “oncofetal FN”, has recently been recognised as a marker of angiogenesis, with its expression being paramount in the hyperplastic endothelia of human glioblastomas.2 On the other hand, several reports have stressed the role of angiogenesis in sustaining growth and invasive potential of malignant tumours. In view of the possible clinical use of radiolabelled monoclonal antibodies (MoAb) able to selectively bind components of the tumorous vasculature as potential angiosuppressive agents for postoperative treatment of cerebral malignancies, angiogenic factors and markers in these tumours are worthy of further study. In this regard, primary anaplastic meningiomas, although relatively rare, represent a challenge, because an aggressive treatment including adjuvant combined modality therapy may affect the poor outcome and warrant palliation of the disease.3 The purpose of the present report was to analyse the distribution of the oncofetal FN in the vessels of a series of human malignant meningiomas and to ascertain whether the occurrence of this isoform may represent a possible basis for postsurgical adjuvant radioimmunotherapy.
Ten primary anaplastic meningiomas (World Health Organisation classification), taken during the course of surgical procedures, were studied. No radiotherapy had been performed before surgery.
Each sample was divided into two portions: one was processed for conventional histopathological diagnosis and the other was snap frozen in liquid nitrogen. Five micron thick cryostat sections were used for immunohistochemical staining after air drying and fixation in absolute cold acetone for 10 minutes. At least three non- consecutive sections were analysed, to evaluate the possible occurrence of false negative results.
Immunostaining was performed using the monoclonal antibody BC-1, specific for the oncofetal FN isoform according to the characterisation performed in this laboratory.1 The antihuman Ki-67 and CD-31 monoclonal antibodies, both purchased from Dako (Carpentaria, CA, USA), were also employed to detect the nuclei of proliferating cells and the vascular endothelium respectively. Double staining experiments were performed according to Sternberger and Shirley.5 The first reaction sequence consisted of the application of the primary monoclonal antibody and incubation with biotinylated goat antimouse IgG (Bio-Spa). Immunoenzymatic staining was then carried out using 3,3-diaminobenzidine tetrahydrochloride (DAB) H202 (Sigma), which yielded a brown reaction product masking antigens and immunoreagents of this first sequence and thus preventing cross binding of the antibodies in the second sequence. Next, the sections were incubated with the second primary monoclonal antibody, then incubated with the secondary antibody and streptavidin-biotinylated alkaline phosphatase complex (Bio-Spa). The red reaction product was obtained using a mixture of 2 mg naphtol AS-MX phosphate (Sigma) dissolved in 200 μl n,n-dimethylformamide (Sigma) and diluted in 9.8 ml 0.1 M Tris-HCI buffer, pH 8.2, and 1 mM levamisole (Sigma). Immediately before use, 10 mg fast-red TR salt (Sigma) were added. Triple staining experiments were performed according to a double staining method, but the first reaction sequence consisted of the application of a mixture of two primary monoclonal antibodies. Gill’s haematoxylin was used as a counterstain, and the sections were mounted in glycergel (Dako). In all examined tumours, most of the vessels, with either thin monostratified or hyperplastic endothelium, were found to express oncofetal FN (figure) Definite staining when using the BC-1 monoclonal antibody approached 100% of the capillary network. The endothelium of larger vessels showed as less ubiquitous staining. However, all but one of the examined tumours exhibited a positive endothelial layer in more than the half of their thin or thick walled vessels. In six out of the 10 anaplastic meningiomas, definite endothelial staining occurred in more than 75% of the larger vessels.
Besides the vascular endothelium, either the stroma or the tumorous cells showed some staining, although highly variable, in individual tumours, both in intensity and in distribution. The degree of the involvement of the neoplastic cells ranged from a few elements scattered in a wholly negative contest, to large cellular areas, without a direct relation with the presence of positive vessels (data not shown). Endothelial cells produce large amounts of FNs in vitro. FN is actually the blend of structurally and functionally different isoforms resulting from the alternative splicing at different regions. The splicing pattern is known to be deregulated in transformed cells.1 The oncofetal B+ FN isoform has been found to occur typically in fetal tissues and in malignancies, being absent in the vessels of adult normal brain.2 Research in this aboratory succeeded in preparing a monoclonal antibody (BC-1) specific for oncofetal FN, an isoform characterised by a peculiar sequence (ED.B) in the central part of its molecule. On the other side, different monoclonal antibodies were prepared, which are either able to recognise all FN isoforms (IST-4), or are specific for FNs that do not contain oncofetal sequence (IST-6).1 A previous study, in which all these monoclonal antibodies were employed showed that, whereas FNs are widely distributed in the endothelia of human gliomas, the occurrence of the oncofetal isoform correlates with the degree of malignancy. Similarly, the expression of the oncofetal FN in the vascular endothelium of benign meningiomas was found to be restricted,2 by contrast with the findings reported here.
Preliminary findings by using the Tc-99m-labelled anti-oncofetal FN monoclonal antibody BC-1, that was administered intravenously, showed that this monoclonal antibody does reach malignant meningiomas in vivo, as well as malignant gliomas, allowing scintigraphic evidence of the tumour and providing a possible basis for vascular targeting.4 In this regard, it can be speculated that administration of tumoricidal radionuclides linked to monoclonal antibody BC-1 might prove to be able to selectively destroy endothelia of meningeal malignancies, while sparing the normal vessels of the brain, and to favourably affect the course of the illness in terms of recurrence rate.
This study was partially supported by the National Research Council (CNR), AIRC, and the Ministry of University and Scientific Research (MURST). We thank Mr Sergio Deseri for his technical help in the preparation of the manuscript.
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