Surgery for gliomas is often confounded by difficulties in distinguishing tumor from surrounding normal brain. For better discrimination, intraoperative optical imaging methods using fluorescent dyes are currently being explored. Understandably, such methods require the demonstration of a high degree of diagnostic accuracy and clinical benefit. Currently, clinical utility is determined by tissue biopsies which are correlated to optical signals, and quantified using measures such as sensitivity, specificity, positive predictive values, and negative predictive values. In addition, surgical outcomes, such as extent of resection rates and/or survival (progression-free survival (PFS) and overall survival (OS)) have been measured. These assessments, however, potentially involve multiple biases and confounders, which have to be minimized to ensure reproducibility, generalizability and comparability of test results. Test should aim at having a high internal and external validity.
A review of the literature was performed for assessing the use of measures of diagnostic accuracy and outcomes of intraoperative optical imaging methods. From these data, Stummer et al., derived recommendations for designing and reporting future studies.
Available literature indicates that potential confounders and biases for reporting the diagnostic accuracy and usefulness of intraoperative optical imaging methods are seldom accounted for. Furthermore, methods for bias reduction are rarely used nor reported.
Detailed, transparent, and uniform reporting on the diagnostic accuracy of intraoperative imaging methods is necessary. In the absence of such reporting, studies will not be comparable or reproducible. Future studies should consider some of the recommendations given here 1).
The use of the optical contrast agent sodium fluorescein (NaFl) to guide resection of gliomas has been under investigation for decades. Although this imaging strategy assumes the agent remains confined to the vasculature except in regions of blood brain barrier (BBB) disruption, clinical studies have reported significant NaFl signal in normal brain tissue, limiting tumor-to-normal contrast. A possible explanation arises from earlier studies, which reported that NaFl exists in both pure and protein-bound forms in the blood, the former being small enough to cross the BBB.
A study of Folaron et al. from the Thayer School of Engineering and Department of Surgery Geisel School of Medicine, Dartmouth College, Hanover; and Section of Neurosurgery, and Norris Cotton Cancer Center, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire, aimed to elucidate the kinetic binding behavior of NaFl in circulating blood and its effect on NaFl accumulation in brain tissue and tumor contrast. Additionally, they examined the blood and tissue kinetics, as well as tumor uptake, of a pegylated form of fluorescein selected as a potential optical analog of gadolinium-based MRI contrast agents.
Cohorts of mice were administered one of the following doses/forms of NaFl: 1) high human equivalent dose (HED) of NaFl, 2) low HED of NaFl, or 3) pegylated form of fluorescein. In each cohort, groups of animals were euthanized 15, 30, 60, and 120 minutes after administration for ex vivo analysis of fluorescein fluorescence. Using gel electrophoresis and fluorescence imaging of blood and brain specimens, the authors quantified the temporal kinetics of bound NaFl, unbound NaFl, and pegylated fluorescein in the blood and normal brain tissue. Finally, they compared tumor-to-normal contrast for NaFl and pegylated-fluorescein in U251 glioma xenografts.
Administration of NaFl resulted in the presence of unbound and protein-bound NaFl in the circulation, with unbound NaFl constituting up to 70% of the signal. While protein-bound NaFl was undetectable in brain tissue, unbound NaFl was observed throughout the brain. The observed behavior was time and dose dependent. The pegylated form of fluorescein showed minimal uptake in brain tissue and improved tumor-to-normal contrast by 38%.
Unbound NaFl in the blood crosses the BBB, limiting the achievable tumor-to-normal contrast and undermining the inherent advantage of tumor imaging in the brain. Dosing and incubation time should be considered carefully for NaFl-based fluorescence-guided surgery (FGS) of glioma. A pegylated form of fluorescein showed more favorable normal tissue kinetics that translated to higher tumor-to-normal contrast. These results warrant further development of pegylated-fluorescein for FGS of glioma 3).
Senders et al., systematically review all clinically tested fluorescent agents for application in FGS for glioma and all preclinically tested agents with the potential for FGS for glioma.
They assessed fluorescent agents by the following outcomes: rate of gross total resection (GTR), overall and progression free survival, sensitivity and specificity in discriminating tumor and healthy brain tissue, tumor-to-normal ratio of fluorescent signal, and incidence of adverse events.
The search strategy resulted in 2155 articles that were screened by titles and abstracts. After full-text screening, 105 articles fulfilled the inclusion criteria evaluating the following fluorescent agents: 5 aminolevulinic acid (5-ALA) (44 studies, including three randomized control trials), fluorescein (11), indocyanine green (five), hypericin (two), 5-aminofluorescein-human serum albumin (one), endogenous fluorophores (nine) and fluorescent agents in a pre-clinical testing phase (30). Three meta-analyses were also identified.
5-ALA is the only fluorescent agent that has been tested in a randomized controlled trial and results in an improvement of GTR and progression-free survival in high-grade gliomas. Observational cohort studies and case series suggest similar outcomes for FGS using fluorescein. Molecular targeting agents (e.g., fluorophore/nanoparticle labeled with anti-EGFR antibodies) are still in the pre-clinical phase, but offer promising results and may be valuable future alternatives. 4).
Mounting evidence suggests that a more extensive surgical resection is associated with an improved life expectancy for both low grade glioma and high grade glioma patients. However, radiographically complete resections are not often achieved in many cases because of the lack of sensitivity and specificity of current neurosurgical guidance techniques at the margins of diffuse infiltrative gliomas. Intraoperative fluorescence imaging offers the potential to improve the extent of resection and to investigate the possible benefits of resecting beyond the radiographic margins.
Liu et al., in 2014 provided a review of wide-field and high-resolution fluorescence-imaging strategies that are being developed for neurosurgical guidance, with a focus on emerging imaging technologies and clinically viable contrast agents. The strengths and weaknesses of these approaches will be discussed, as well as issues that are being addressed to translate these technologies into the standard of care 5).
322 patients aged 23-73 years with suspected malignant glioma amenable to complete resection of contrast-enhancing tumour were randomly assigned to 20 mg/kg bodyweight 5-aminolevulinic acid for fluorescence-guided resection (n=161) or to conventional microsurgery with white light (n=161). The primary endpoints were the number of patients without contrast-enhancing tumour on early MRI (ie, that obtained within 72 h after surgery) and 6-month progression-free survival as assessed by MRI. Secondary endpoints were volume of residual tumour on postoperative MRI, overall survival, neurological deficit, and toxic effects. We report the results of an interim analysis with 270 patients in the full-analysis population (139 assigned 5-aminolevulinic acid, 131 assigned white light), which excluded patients with ineligible histological and radiological findings as assessed by central reviewers who were masked as to treatment allocation; the interim analysis resulted in termination of the study as defined by the protocol. Primary and secondary endpoints were analysed by intention to treat in the full-analysis population. The study is registered at http://www.clinicaltrials.gov as NCT00241670.
FINDINGS: Median follow-up was 35.4 months (95% CI 1.0-56.7). Contrast-enhancing tumour was resected completely in 90 (65%) of 139 patients assigned 5-aminolevulinic acid compared with 47 (36%) of 131 assigned white light (difference between groups 29% [95% CI 17-40], p<0.0001). Patients allocated 5-aminolevulinic acid had higher 6-month progression free survival than did those allocated white light (41.0% [32.8-49.2] vs 21.1% [14.0-28.2]; difference between groups 19.9% [9.1-30.7], p=0.0003, Z test). Groups did not differ in the frequency of severe adverse events or adverse events in any organ system class reported within 7 days after surgery.
INTERPRETATION: Tumour fluorescence derived from 5-aminolevulinic acid enables more complete resections of contrast-enhancing tumour, leading to improved progression-free survival in patients with malignant glioma 6).