Quantitative fluorescence using 5-aminolevulinic acid-induced protoporphyrin IX biomarker as a surgical adjunct in low-grade glioma surgery

Abstract

Object: Previous studies in high-grade gliomas (HGGs) have indicated that protoporphyrin IX (PpIX) accumulates in higher concentrations in tumor tissue, and, when used to guide surgery, it has enabled improved resection leading to increased progression-free survival. Despite the benefits of complete resection and the advances in fluorescence-guided surgery, few studies have investigated the use of PpIX in low-grade gliomas (LGGs). Here, the authors describe their initial experience with 5-aminolevulinic acid (ALA)-induced PpIX fluorescence in a series of patients with LGG.

Methods: Twelve patients with presumed LGGs underwent resection of their tumors after receiving 20 mg/kg of ALA approximately 3 hours prior to surgery under an institutional review board-approved protocol. Intraoperative assessments of the resulting PpIX emissions using both qualitative, visible fluorescence and quantitative measurements of PpIX concentration were obtained from tissue locations that were subsequently biopsied and evaluated histopathologically. Mixed models for random effects and receiver operating characteristic curve analysis for diagnostic performance were performed on the fluorescence data relative to the gold-standard histopathology.

Results: Five of the 12 LGGs (1 ganglioglioma, 1 oligoastrocytoma, 1 pleomorphic xanthoastrocytoma, 1 oligodendroglioma, and 1 ependymoma) demonstrated at least 1 instance of visible fluorescence during surgery. Visible fluorescence evaluated on a specimen-by-specimen basis yielded a diagnostic accuracy of 38.0% (cutoff threshold: visible fluorescence score ≥ 1, area under the curve = 0.514). Quantitative fluorescence yielded a diagnostic accuracy of 67% (for a cutoff threshold of the concentration of PpIX [CPpIX] > 0.0056 μg/ml, area under the curve = 0.66). The authors found that 45% (9/20) of nonvisibly fluorescent tumor specimens, which would have otherwise gone undetected, accumulated diagnostically significant levels of CPpIX that were detected quantitatively.

Conclusions: The authors' initial experience with ALA-induced PpIX fluorescence in LGGs concurs with other literature reports that the resulting visual fluorescence has poor diagnostic accuracy. However, the authors also found that diagnostically significant levels of CPpIX do accumulate in LGGs, and the resulting fluorescence emissions are very often below the detection threshold of current visual fluorescence imaging methods. Indeed, at least in the authors' initial experience reported here, if quantitative detection methods are deployed, the diagnostic performance of ALA-induced PpIX fluorescence in LGGs approaches the accuracy associated with visual fluorescence in HGGs.

Keywords: 5-aminolevulinic acid; ALA = 5-aminolevulinic acid; AUC = area under the curve; CPpIX = concentration of PpIX; HGG = high-grade glioma; LGG = low-grade glioma; NPV = negative predictive value; PPV = positive predictive value; PpIX = protoporphyrin IX; ROC = receiver operating characteristic; biomedical optics; brain tumor; fluorescence-guided surgery; low-grade glioma; oncology; optical spectroscopy; protoporphyrin IX; quantitative fluorescence.

Figures

Fig. 1

Intraoperative fluorescence in 6 LGGs interrogated with the quantitative probe: CPpIX in specimens histologically categorized as either tumor negative, (−) Tumor, or tumor positive, (+) Tumor, with no visible fluorescence, (-F), or positive visible fluorescence, (+F). Nonvisibly fluorescent tumor tissues (45%, 9/20 specimens) accumulated levels of CPpIX (cutoff value > 0.0056 μg/ml) that were not identified with intraoperative visible fluorescence imaging. The one false positive for visible fluorescence (-Tumor, +F) was from a sample of brightly visibly fluorescent normal hippocampus.

Fig. 2

Intraoperative fluorescence using the quantitative probe in LGG subtypes: CPpIXin specimens categorized as either tumor negative, (−) Tumor, or tumor positive, (+) Tumor, with no visible fluorescence, (-F), or positive visible fluorescence, (+F), in each category. Nine data points with CPpIX < 0.001 μg/ml are not displayed: oligoastrocytoma: 3 (-F, -Tumor) and 3 (-F, +Tumor); oligodendroglioma: 1 (-F, -Tumor) and 1 (-F, +Tumor); and ganglioglioma: 1 (-F, +Tumor).

Fig. 3

Intraoperative quantitative and qualitative measurements of PpIX during 2 oligoastrocytoma surgical cases with differing visible fluorescence characteristics. Normal cortex under white light (A) , violet-blue light excitation demonstrating no visible fluorescence (B), neuronavigation coordinates at the cortical surface far removed from the area of T2 hyperintensity (C), and quantitative spectroscopic probe measurements demonstrating a typical autofluorescence spectrum without signs of the signature PpIX main peak at 635 nm (D). Surgical cavity of one oligoastrocytoma under white light (E), violet-blue light excitation with no visible fluorescence (F), neuronavigation coordinates at the T2 hyperintensity region within the tumor bulk (G), and quantitative spectroscopic probe measurements (H) with the typical PpIX main peak at 635 nm and approximately 50 times more PpIX than normal cortex, and again near the end of surgery under white light (I), violet-blue light excitation with no visible fluorescence (J), neuronavigation coordinates at the T2 hyperintensity preoperative MRI border (K), and quantitative spectroscopic probe measurements with the typical PpIX main peak at 635 nm and approximately 150 times more PpIX than normal cortex (L). Surgical cavity of another oligoastrocytoma that demonstrated visible fluorescence under white light (L), violet-blue light excitation illustrating visible fluorescence (N), neuronavigation coordinates within the tumor bulk near a region of contrast enhancement on T1-weighted MRI (O), and quantitative spectroscopic probe measurements (P) of a spectrum with the typical PpIX main peak at 635 nm and 710 nm and approximately 10,000 times more PpIX than normal cortex.