gms | German Medical Science

Objective: The standard of care for diagnosis and therapy monitoring of gliomas is magnetic resonance imaging (MRI) which, however, provides only an indirect and incomplete representation of the tumor mass, offers limited information for patient stratification according to WHO-grades and may insufficiently indicate tumor relapse after anti-angiogenic therapy. In order to develop a molecular imaging approach facilitating the identification of malignant gliomas and improving therapy monitoring we investigated Anticalins as PET-tracers directed against the oncofetal fibronectin carrying the extra domain B (ED-B), which is an extracellular matrix component in neoplastic tissue and newly forming blood-vessels.

Method: Anticalins are alternative binding proteins generated via combinatorial protein design from the human lipocalin scaffold, which offer novel diagnostic reagents for histology and imaging applications. Here, the Anticalins N7A, N7E and N9B possessing exquisite affinity and specificity for ED-B were used for immunohistochemical studies, alone or together with immuno-detection of endothelia and fibronectin, in biopsies from 41 patients with confirmed gliomas of WHO-grade I to IV, or in non-neoplastic brain samples. Anticalins were also used to detect ED-B in live cells by FACS, and autoradiography of human primary Glioblastoma multiforme (GBM) specimen was performed with the radiolabeled version 123I-N7A. PET-based detection of gliomas in vivo was investigated in an orthotopic, immunocompetent mouse model with 89Zr-N7E.

Results: Anticalins specifically detect ED-B in GBM but not in tumors of lower histopathological grade or in tumor-free brain. In primary GBM samples, ED-B specific Anticalins locate to fibronectin-rich perivascular areas that are associated with angiogenesis. Anticalins specifically detect ED-B both in fixed tumor specimen and on live cells, as evidenced by cytofluorometry. Beyond that, 123I-N7A demonstrated specific binding in human GBM-tissue samples and 89Zr-N7E was successfully applied to diagnose gliomas in vivo in a preclinical mouse model.

Conclusions: ED-B is specifically expressed in malignant gliomas but not elsewhere in the brain. Anticalins directed against ED-B, novel engineered non-Ig binding proteins, are useful PET-tracers to diagnose glioblastomas and show potential to improve the classification and localization of primary brain tumors by molecular imaging approaches in the future.