The value of visible 5-ALA fluorescence and quantitative protoporphyrin IX analysis for improved surgery of suspected low-grade gliomas

Abstract

Objective: In patients with suspected diffusely infiltrating low-grade gliomas (LGG), the prognosis is dependent especially on extent of resection and precision of tissue sampling. Unfortunately, visible 5-aminolevulinic acid (5-ALA) fluorescence is usually only present in high-grade gliomas (HGGs), and most LGGs cannot be visualized. Recently, spectroscopic probes were introduced allowing in vivo quantitative analysis of intratumoral 5-ALA-induced protoporphyrin IX (PpIX) accumulation. The aim of this study was to intraoperatively investigate the value of visible 5-ALA fluorescence and quantitative PpIX analysis in suspected diffusely infiltrating LGG.

Methods: Patients with radiologically suspected diffusely infiltrating LGG were prospectively recruited, and 5-ALA was preoperatively administered. During resection, visual fluorescence and absolute tissue PpIX concentration (CPpIX) measured by a spectroscopic handheld probe were determined in different intratumoral areas. Subsequently, corresponding tissue samples were safely collected for histopathological analysis. Tumor diagnosis was established according to the World Health Organization 2016 criteria. Additionally, the tumor grade and percentage of tumor cells were investigated in each sample.

Results: All together, 69 samples were collected from 22 patients with histopathologically confirmed diffusely infiltrating glioma. Visible fluorescence was detected in focal areas in most HGGs (79%), but in none of the 8 LGGs. The mean CPpIX was significantly higher in fluorescing samples than in nonfluorescing samples (0.693 μg/ml and 0.008 μg/ml, respectively; p < 0.001). A significantly higher mean percentage of tumor cells was found in samples with visible fluorescence compared to samples with no fluorescence (62% and 34%, respectively; p = 0.005), and significant correlation of CPpIX and percentage of tumor cells was found (r = 0.362, p = 0.002). Moreover, high-grade histology was significantly more common in fluorescing samples than in nonfluorescing samples (p = 0.001), whereas no statistically significant difference in mean CPpIX was noted between HGG and LGG samples. Correlation between maximum CPpIX and overall tumor grade was highly significant (p = 0.005). Finally, 14 (40%) of 35 tumor samples with no visible fluorescence and 16 (50%) of 32 LGG samples showed significantly increased CPpIX (cutoff value: 0.005 μg/ml).

Conclusions: Visible 5-ALA fluorescence is able to detect focal intratumoral areas of malignant transformation, and additional quantitative PpIX analysis is especially useful to visualize mainly LGG tissue that usually remains undetected by conventional fluorescence. Thus, both techniques will support the neurosurgeon in achieving maximal safe resection and increased precision of tissue sampling during surgery for suspected LGG.Clinical trial registration no.: NCT01116661 (clinicaltrials.gov).

Keywords: 5-ALA = 5-aminolevulinic acid; CE = contrast enhancement; CPpIX = PpIX concentration; DTI = diffusion tensor imaging; HGG = high-grade glioma; IDH = isocitrate dehydrogenase; LGG = low-grade glioma; MRI = magnetic resonance imaging; NPV = negative predictive value; PPV = positive predictive value; PpIX = protoporphyrin IX; PpIX analysis; ROC = receiver operating characteristic; San Francisco; UCSF = University of California; WHO = World Health Organization; anaplastic foci; oncology; suspected LGG; tumor visualization; visible fluorescence.

Figures

FIG. 1.

Visible 5-ALA fluorescence and quantitative PpIX analysis in tissue samples of suspected LGGs. A: The median percentage of tumor cells was significantly higher (asterisks) in tissue samples with visible fluorescence than in those with no fluorescence. B: Glioma tissue with high-grade histology (WHO grades III and IV) was significantly more common in fluorescing samples than in nonfluorescing samples. C: The median CPpIX was significantly higher in suspected tumor regions than in suspected margin areas. D: The median CPpIX was significantly higher in fluorescing samples than in nonfluorescing samples. E: Percentage of tumor cells and CPpIX showed a significant correlation. F: No statistically significant difference in the median CPpIX between LGG and HGG tissue samples was noted. G: With the assistance of ROC curve analysis an optimal CPpIX cutoff value (0.005 μg/ml) was defined to distinguish between tumor and tumor-free/normal tissue in our study. The boxplots indicate interquartile ranges (boxes) with whiskers extending to 1.5 times the height of the box or to the minimum or maximum values (SPSS default).

FIG. 2.

Illustrative case of a patient with a newly diagnosed suspected LGG in the right frontal lobe with final histology of an oligodendroglioma WHO grade II (case 6). A and B: No CE is detected within the tumor by T1-weighted MRI sequences (A) and the lesion is hyperintense on FLAIR images (B). C–E: The first analyzed area inside the tumor verified by neuronavigation (C) shows only slight macroscopic abnormalities under white-light microscopy (D) and this tissue cannot be visualized by visible fluorescence using violet-blue excitation light (E). F: Quantitative PpIX analysis reveals significantly increased levels of CPpIX of 0.013 μg/ml in this region and histology of this sample corresponds to LGG tissue. G–I: The second area at the suspected tumor margin (G) appears to be “normal” under white-light microscopy (H) and no visible fluorescence is detected (I). J: Quantitative PpIX analysis shows a very low CPpIX of < 0.001 μg/ml in this region and histology does not reveal tumor tissue in the corresponding tissue sample. Interestingly, an additional shifted peak at 627 nm is present aside from the typical main PpIX peak at 635 nm in this PpIX analysis. K–M: In the area of normal cortex (K), no abnormalities under white-light (L) and no visible fluorescence (M) are present. N: Quantitative PpIX analysis shows a very low CPpIX of 0.001 μg/ml in the region of normal cortex.

FIG. 3.

Illustrative case of a patient with a recurrent suspected LGG in the left frontal lobe with final histology of an astrocytoma WHO grade III (case 3). A and B: Focal CE is found within the tumor by T1-weighted MRI sequences (A) and the lesion is hyperintense on FLAIR images (B). C–E: The first area inside the nonenhancing tumor (C) shows only slight macroscopic abnormalities under white-light microscopy (D) and this tissue cannot be visualized by visible fluorescence with assistance of violet-blue excitation light (E). F: Quantitative PpIX analysis reveals significantly increased levels of CPpIX of 0.035 μg/ml in this region and histology of this sample corresponds to LGG tissue. G–I: The second area inside the tumor near the region of focal CE shows macroscopic abnormalities comparable to the first analyzed area under white-light microscopy (H) but shows a small region of visible fluorescence (I). J: Quantitative PpIX analysis of this small fluorescing region shows a very high CPpIX of 0.216 μg/ml, and histology reveals tissue of an HGG in the corresponding sample. K–M: In the area of normal cortex (K), no abnormalities are seen under white-light (L), and no visible fluorescence is present (M). N: Quantitative PpIX analysis shows a very low CPpIX of < 0.001 μg/ml in the region of normal cortex.