Complementary Molecular and Elemental Mass-Spectrometric Imaging of Human Brain Tumors Resected by Fluorescence-Guided Surgery

Abstract

Fluorescence-guided surgery (FGS) has been established as a powerful technique for glioblastoma resection. After oral application of the prodrug 5-aminolevulinic acid (5-ALA), protoporphyrin IX (PpIX) is formed as an intermediate of the heme-biosynthesis cascade and accumulates within the tumor. By intraoperative fluorescence microscopy, the specific PpIX fluorescence can be used to differentiate the tumor from healthy brain tissue. To investigate possible limitations of fluorescence diagnosis, the complementary use of molecular and elemental mass-spectrometry imaging (MSI) is presented. Matrix-assisted laser-desorption-ionization mass spectrometry (MALDI-MS) is used to examine the distribution of PpIX and heme b in human brain tumors. MALDI-MS/MS imaging is performed to validate MS data and improve the signal-to-noise ratio (S/N). Comparing the imaging results with histological evaluation, increased PpIX accumulation in areas of high tumor-cell density is observed. Heme b accumulation are only found in areas of blood vessels and hemorrhage, confirming the hampered transformation from PpIX to heme b in glioblastoma tissue. Investigation of non-neoplastic brain tissue and glioblastoma resected without external 5-ALA administration as control samples with true-negative fluorescence verified the absence of PpIX accumulation. Analysis of necrotic tumor tissue and gliosarcoma, one rare type of glioma appearing nonfluorescent during FGS, as case examples with false-negative-fluorescence diagnosis, revealed the absence of significant amounts of PpIX, indicating an impairment of PpIX formation. Molecular analysis is complemented by quantitative laser ablation-inductively coupled plasma (LA-ICP) MSI correlating heme b and Fe distribution. Mathematical pixel-by-pixel correlation of molecular and elemental data revealed a positive correlation with heteroscedasticity for the spatially resolved heme b signal intensities and Fe concentrations.

Spectral complexity of 5-ALA induced PpIX fluorescence in guided surgery: a clinical study towards the discrimination of healthy tissue and margin boundaries in high and low grade gliomas

“…Quantification of PpIX concentrations is more sensitive than fluorescence microscopy but also shows sensitivity issues in low density tumor cells regions in HGG [28] or LGG [29].These techniques rely on the assumption that the emitted PpIX fluorescence intensity is proportional to the concentration of PpIX. This is supported by the link between the fluorescence emitted intensity and the tumor cellular density [33,34]. However, some studies show that the microenvironment could have an important role in the intensity of fluorescence.…”

“…Quantification techniques rely on the assumption that the emitted PpIX fluorescence intensity is proportional to the concentration of PpIX. This is supported by the link between the fluorescence emitted intensity and the tumor cellular density [17,33,34]. However, some studies showed more complex links between the PpIX concentration and the shape of fluorescence spectra.…”

Mass Spectrometry for Cancer Biomarkers

“…Although neuronavigational guidance by MRI or positron emission tomography (PET) aids in localizing the tumor tissue, the brain shift and tissue deformation may negatively influence the accuracy of resection [94,95]. An option to overcome these limitations is the application of intraoperative imaging; however, besides being expensive they increase the surgery time [95].…”

“…Since the introduction of fluorescence-guided surgery (FGS) in 1998 by Stummer et al subsequent to proagent 5-aminolevulinic acid (5-ALA) treatment, contrast enhancement by a fluorescent dye has progressed and represents today an important and powerful technique in GBM resection [94,95]. After oral application of 5-ALA approximately 3 h before anesthesia, in a dose of 20 mg/kg body weight, the small molecule crosses the abnormal blood-brain barrier into the peritumoral tissue and accumulates within the tumor.…”

“…As a precursor of heme B, in an intermediate step of the heme biosynthesis cascade, 5-ALA is converted to protoporphyrin IX, a red fluorescent at 635 nm upon blue light excitation. Since high-grade gliomas are known to be characterized by augmented cell proliferation, tumor cell density and Mass Spectrometry for Cancer Biomarkers DOI: http://dx.doi.org/10.5772/intechopen.85609 microvessel density, the greater fluorescence intensities generated by accumulation of high cellular PpIX concentrations, improve differentiation of tumor from healthy brain tissue during intraoperative fluorescence microscopy [95][96][97]. Nevertheless, in some cases, false-positive and false-negative fluorescence results cannot be excluded.…”