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Vol. 22, Art. 40 (pp. 571-593)    |    2021       

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Morphological characteristics of the perifocal zone of glioblastoma using radiation, immunohostochemical and immunofluorescent methods

Galkovsky B.E., Mitrofanova L.B., Ryzhkova D.V., Sulin K.A., Gulyaev D.A., Danilova I.A., Sidorin V.S., Gaikova O.N.

Almazov National Medical Research Centre

Brief summary

Glioblastoma (GBM) is a malignant brain tumor with high resistance to radio- and chemotherapy. Patient survival rates remain low, with average life expectancy after diagnosis being 13-15 months. Radical surgical resection is one of the main aspects of GBM treatment. As before, the questions of determining the boundaries, morphological composition and biological potential of the GBM perifocal zone remain open. The aim of the study was to study the expression of FoxM1 and Ki-67, as well as the fluorescence of protoporphyrin IX in the perifocal zone of the GBM, in comparison with the data of radiation methods. Materials and methods: 10 primary GBM, 10 samples of their perifocal zone, 10 fragments of a normal brain (control group) were studied. Before surgery, patients were prescribed oral administration of 5-aminolevulinic acid. The material was taken from the zone of red (GBM) and pink fluorescence. In all cases, histological, immunohistochemical analysis with antibodies to FoxM1 and Ki-67, and morphometry were carried out on paraffin sections. Using confocal laser scanning microscopy (CLSM), smears-prints, paraffin sections, frozen sections, native material of GBM and its perifocal growth zone were examined by the CLSM method with staining of cell nuclei with DAPI and fluorescent trackers of mitochondria, reactive oxygen species, and lysosomes. Statistical analysis of the data obtained was carried out at a confidence level of p = 0.05. Results: the histological structure of the GBM perifocal zone varied from an almost normal brain to the morphological picture of diffuse astrocytoma. In the perifocal zone of GBM, the level of proliferative activity according to Ki-67 was 5.6 [1.8; 18.0] %, which was noticeably lower (U = 3.0; significance 0.0001) of the level of proliferative activity of tumor cells - 33.1 [22.9; 37.7]%. The level of FoxM1 expression was found to be high both in the perifocal zone (U = 43.5; significance 0.631) and in the tumor zone: 96.5 [73.3; 100] % and 100 [75.3; 100]% respectively. With CLSM of native preparations of GBM tissue and perifocal zone, background red fluorescence of protoporphyrin IX (PrpIX) was observed in the cytoplasm of cells, in the lumen and walls of blood vessels. Also, colocalization of the PrpIX signal and lysosomes, mitochondria, and reactive oxygen species was found. Conclusions: a high level of FoxM1 expression was found in the cells of the GBM perifocal zone, and Ki-67 values corresponded to grade 2-4 gliomas. PrpIX was found in the mitochondria of tumor cells, their lysosomes, not only in the tumor, but also in the perifocal zone and vessels. The data obtained indicate the presence of the tumor potential of the perifocal zone of the tumor, and are the basis for recommending its surgical resection.


Key words

glioblastoma, confocal scanning laser microscopy, perifocal zone, FoxM1

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