Fluorescence-Guided Resection of Malignant Glioma with 5-ALA

Abstract

Malignant gliomas are extremely difficult to treat with no specific curative treatment. On the other hand, photodynamic medicine represents a promising technique for neurosurgeons in the treatment of malignant glioma. The resection rate of malignant glioma has increased from 40% to 80% owing to 5-aminolevulinic acid-photodynamic diagnosis (ALA-PDD). Furthermore, ALA is very useful because it has no serious complications. Based on previous research, it is apparent that protoporphyrin IX (PpIX) accumulates abundantly in malignant glioma tissues after ALA administration. Moreover, it is evident that the mechanism underlying PpIX accumulation in malignant glioma tissues involves an abnormality in porphyrin-heme metabolism, specifically decreased ferrochelatase enzyme activity. During resection surgery, the macroscopic fluorescence of PpIX to the naked eye is more sensitive than magnetic resonance imaging, and the alert real time spectrum of PpIX is the most sensitive method. In the future, chemotherapy with new anticancer agents, immunotherapy, and new methods of radiotherapy and gene therapy will be developed; however, ALA will play a key role in malignant glioma treatment before the development of these new treatments. In this paper, we provide an overview and present the results of our clinical research on ALA-PDD.

Figures

Figure 1

The simplified synthetic pathway of ALA-induced PpIX. ALA is an endogenous substance. It is incorporated into the mitochondria, repeatedly transported between the cytoplasm and the mitochondria, and finally used for heme synthesis. ALA is converted into porphobilinogen by the enzyme ALA dehydratase, and porphobilinogen is sequentially converted into hydroxymethylbilane, uroporphyrinogen III, coproporphyrinogen III, protoporphyrinogen IX, and finally PpIX by the enzyme protoporphyrinogen oxidase. The final product heme is synthesized from PpIX and iron (Fe) by the enzyme ferrochelatase (FeC). This biosynthetic pathway is considered to be the same in all organisms (PENTA: pentacarboxyl porphyrin, COPRO I or III: coproporphyrin I or III, and HARDERO: harderoporphyrin).

Figure 2

The amount of porphyrins in the control brain tissue and glioblastoma tissue. The white bar shows control tissue from a brain tumor patient (n = 14), and the gray bar shows glioblastoma (n = 28) (∗p < 0.05) (PENTA: pentacarboxyl porphyrin, COPRO I or III: coproporphyrin I or III, HARDERO: harderoporphyrin, and PpIX: protoporphyrin IX).

Figure 3

(a) Ferrochelatase (FeC) activity. Note that the FeC activity clearly decreased more in the malignant glioma cells (n = 28) than in the control cells (n = 14) (∗p < 0.01). The FeC activity is expressed by the amount of heme biosynthesized from PpIX per unit time. Namely, the unit of FeC activity is nmol of heme formed/mg/organ/h. The unit of PpIX is nmol/g/organ. (b) The correlation between the FeC activity and the amount of PpIX in the mitochondria or in the whole cell. The left panel shows the relationship between the total intracellular amount of PpIX and the FeC activity. The right panel shows the relationship between the amount of PpIX in the mitochondria and the FeC activity. Note that the FeC activity and the total amount of PpIX were positively correlated (R = 0.94). The FeC activity and the amount of PpIX in the mitochondria were negatively correlated (R = −0.412). Namely, the FeC activity decreased with an increase in the accumulated amount of PpIX in the mitochondria. (c) The amount of heme in the mitochondria. The amount of heme in the mitochondria was compared between the control cells (n = 14) and tumor cells (n = 28). This amount was remarkably smaller in the tumor tissue than in the normal tissue (∗p < 0.01).

Figure 4

(a) An intraoperative photograph of the brain surface. (A) The tumor surface under xenon white light. Hypervascularity is observed; however, the tumor boundary is vague. (B) By irradiating excitation light of violet-blue, red fluorescence from the tumor tissues is observed and the tumor boundary is clearly recognized. (C) In the cavity after tumor removal under xenon white light, the residual tumor is not clearly recognized. (D) In the same operative field as (C) under excitation light of violet-blue, the residual tumor is clearly recognized. (b) The R/G ratio with fluorescence spectrum. We obtained the amplitude of the red (R) PpIX fluorescence from the glioma tissues and the green (G) autofluorescence from the glioma tissues in a spectrum.

Figure 5

The amount of PpIX accumulation in each grade of glioma. High-grade malignant glioma contains a high concentration of PpIX in the tissue. This sign of (−)~(+++) shows the intensity of fluorescence to the naked eye.

Figure 6

The size of the tumor using different methods. (a) MRI image shows the glioblastoma in the right temporal lobe. (b) It was 24 mm on enhanced MRI. (c) It was 20 mm on macroscopic observation under the white light. (d) It was 30 mm on macroscopic observation and fluorescence to the naked eye.

Figure 7

The analysis of the fluorescence spectrum and the histology in each lesion. Lesion (a) was central necrosis without macroscopic fluorescence. Lesion (b) showed the highest fluorescence macroscopically. Lesion (c) showed a faint fluorescence macroscopically. Lesion (d), adjacent to lesion (c), did not show macroscopic fluorescence. Lesion (e) did not show any fluorescence. The left panel shows the analysis of the fluorescence spectrum, and the right panel shows the histological examinations and MIB-1 index.