Intraoperative fluorescence imaging allows real-time identification of diseased tissue during surgery without being influenced by brain shift and surgery interruption. 5-Aminolevulinic acid, useful for malignant gliomas and other tumors, is the most broadly explored compound approved for fluorescence-guided resection. Intravenous fluorescein sodium has recently received attention, highlighting tumor tissue based on extravasation at the blood-brain barrier (defective in many brain tumors). Fluorescein in perfused brain, unselective extravasation in brain perturbed by surgery, and propagation with edema are concerns. Fluorescein is not approved but targeted fluorochromes with affinity to brain tumor cells, in development, may offer future advantages.
Key points
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Intraoperative fluorescence imaging enables real-time intraoperative identification of diseased brain tissue, which can be exploited for resection. As a tool, it expands the existing armamentarium but does not obviate immaculate surgical technique, a profound understanding of surgical anatomy, and the copious use of mapping and monitoring techniques.
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5-Aminolevulinic acid is approved for intraoperative fluorescence imaging in many countries of the world.
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This compound is nonfluorescent and is converted to the fluorescent moiety PPIX within the tumor cell. A large body of literature supports its use in gliomas but also in other neurosurgically relevant tumors, for example, meningiomas.
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Fluorescein sodium, an older compound, has recently been receiving new attention. Fluorescein is extravasated at the breached blood-brain barrier, thus marking areas of blood-brain barrier breakdown, which are associated with brain tumors. The potential and pitfalls of this approach are subject to ongoing investigations. The regulatory status is unresolved.
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Novel, targeted fluorochromes are in preclinical and early clinical development and may provide high selectivity and high fluorescence yields in the future.
Video content accompanies this article at http://www.neurosurgery.theclinics.com .
Introduction
It is commonly acknowledged that even experienced neurosurgeons, using the optical information provided by the surgical microscope, their tactile sense, and a profound knowledge of anatomy, will not always be able to discriminate normal brain from brain infiltrated by tumor. This is of importance in malignant gliomas for which it is now well accepted that safe maximal resections are of benefit for both the rates of recurrence and the life expectancy of the patients. It was many years ago that Albert and colleagues, 1994, first demonstrated conclusively how strongly neurosurgeons tend to overestimate the degree of resection in malignant gliomas when relying on optical and tactile senses alone by correlating the surgeon’s impression of radicality with early postoperative MRI. Neuronavigation, introduced during the early 1990s, was certainly helpful in overcoming this problem but suffers from the limitation of brain shift and frequent interruptions of surgery for orientation. Intraoperative imaging using the MRI was the next game changer (see Ganesh Rao article,“ Intraoperative MRI and Maximizing Extent of Resection ”, in this issue). Even though the value of intraoperative MRI is undisputed, the high price of dedicated systems and the additional effort required for obtaining images still limits the distribution of such systems into many neurosurgical wards. The ideal technique for intraoperative identification of tissue would provide real-time tissue information during the actual process of resection, without the worries related to brain shift, and still be affordable. Intraoperative tissue fluorescence has the power to fulfill these requirements. With diseased tissue showing fluorescence in contradistinction to nondiseased tissue, and fluorescence being made visible to the surgeon, the surgeon can potentially operate directly on the tissue. It must be remembered, however, that any form of tissue staining in the brain does not diminish the responsibility of the surgeon to carefully reflect on whether to resect and to minimize risks to patients, optimally using state-of-the-art mapping and monitoring technology. Fluorescence also does not obviate an intimate knowledge of surgical anatomy while respecting microsurgical principles of resection or wise selection of patients. See Box 1 for advantages and disadvantages of fluorescence imaging.
Fluorescence is a tool and not a therapy. The information from tissue fluorescence needs to be used wisely, while respecting principles of case selection, microsurgery, and mapping and monitoring of neurologic function.
Advantages
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Real-time information
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Information provided through the surgical microscope with accustomed magnification
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Information provided from tissue and not from image
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No concern for brain shift
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Can be repeated as often as necessary
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Depending on type of fluorescence extended parts of the surgery can be performed using fluorescence only
Drawbacks
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Only 2-dimensional information
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Fluorescence can be obscured by overhanging tissue, blood, hemostatic agents
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Fluorophores can be bleached or destroyed by coagulation
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Requires additional drugs in many cases; regulatory issues
Introduction
It is commonly acknowledged that even experienced neurosurgeons, using the optical information provided by the surgical microscope, their tactile sense, and a profound knowledge of anatomy, will not always be able to discriminate normal brain from brain infiltrated by tumor. This is of importance in malignant gliomas for which it is now well accepted that safe maximal resections are of benefit for both the rates of recurrence and the life expectancy of the patients. It was many years ago that Albert and colleagues, 1994, first demonstrated conclusively how strongly neurosurgeons tend to overestimate the degree of resection in malignant gliomas when relying on optical and tactile senses alone by correlating the surgeon’s impression of radicality with early postoperative MRI. Neuronavigation, introduced during the early 1990s, was certainly helpful in overcoming this problem but suffers from the limitation of brain shift and frequent interruptions of surgery for orientation. Intraoperative imaging using the MRI was the next game changer (see Ganesh Rao article,“ Intraoperative MRI and Maximizing Extent of Resection ”, in this issue). Even though the value of intraoperative MRI is undisputed, the high price of dedicated systems and the additional effort required for obtaining images still limits the distribution of such systems into many neurosurgical wards. The ideal technique for intraoperative identification of tissue would provide real-time tissue information during the actual process of resection, without the worries related to brain shift, and still be affordable. Intraoperative tissue fluorescence has the power to fulfill these requirements. With diseased tissue showing fluorescence in contradistinction to nondiseased tissue, and fluorescence being made visible to the surgeon, the surgeon can potentially operate directly on the tissue. It must be remembered, however, that any form of tissue staining in the brain does not diminish the responsibility of the surgeon to carefully reflect on whether to resect and to minimize risks to patients, optimally using state-of-the-art mapping and monitoring technology. Fluorescence also does not obviate an intimate knowledge of surgical anatomy while respecting microsurgical principles of resection or wise selection of patients. See Box 1 for advantages and disadvantages of fluorescence imaging.
Fluorescence is a tool and not a therapy. The information from tissue fluorescence needs to be used wisely, while respecting principles of case selection, microsurgery, and mapping and monitoring of neurologic function.
Advantages
- •
Real-time information
- •
Information provided through the surgical microscope with accustomed magnification
- •
Information provided from tissue and not from image
- •
No concern for brain shift
- •
Can be repeated as often as necessary
- •
Depending on type of fluorescence extended parts of the surgery can be performed using fluorescence only
Drawbacks
- •
Only 2-dimensional information
- •
Fluorescence can be obscured by overhanging tissue, blood, hemostatic agents
- •
Fluorophores can be bleached or destroyed by coagulation
- •
Requires additional drugs in many cases; regulatory issues
Fluorescence: theoretic background
In general, fluorescence is a characteristic of many dyes that, when illuminated by light with a short wave-length, emit light of a longer wavelength. By using appropriate filters, the emission light can be excluded from the observer, allowing only the emitted fluorescence light to become visible. Thus, the observer can perceive the distribution of the dye selectively. If the dye is associated with tumor tissue, the distribution of otherwise not clearly visible tumor can be perceived based on tissue fluorescence.
In general, 4 types of approaches to intraoperative fluorescence can be distinguished ( Table 1 ). Tissue fluorescence based on passive permeability, for example, indocyanine green (ICG) or fluorescein; tissue fluorescence induced by specific metabolic characteristics (eg, 5-aminolevulinic acid [ALA]); autofluorescence; and, finally, fluorescence derived by fluorescent probes targeting or being retained by brain tumor tissue. These methods differ much in their background, selectivity, stage of clinical development, and versatility. In general, when evaluating these methods and to allow a comparison, several aspects need to be kept in mind:
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The selectivity of accumulation
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Time dependency of signal and signal strength
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Visibility of the fluorochrome to the surgeon
Technique | Basis | Regulatory Ramifications | Developmental Status | Publications |
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ALA | Metabolic | Approval by drug or device laws in Europe and other countries | Widespread, human use | |
ICG | Passive permeability | Drug, device; approved for intraoperative angiography, not approved for fluorescence-guided resection | Human use, off label | |
Fluorescein | Passive permeability | Drug, device, not approved for fluorescence-guided resection | Human use, strictly in studies | |
Alkylphosphocholine | Targeted | Drug, device, not approved for fluorescence-guided resection | Human use phase I–II | |
Tumor paint (chlorotoxin-infrared-dye conjugate) | Targeted | Drug, device not approved for fluorescence-guided resection | In vitro, in vivo | |
Antibodies against tumor epidermal growth factor receptor coupled to near infrared dye | Targeted | Drug, device not approved for fluorescence-guided resection | In vitro, in vivo | |
Phospholipid nanoparticles containing near infrared dye | Targeted | Drug, device not approved for fluorescence-guided resection | In vivo | |
Autofluorescence | Intrinsic | Device | In vivo, clinical |
Selectivity of accumulation
Selectivity of tumor delineation, of course, is a crucial issue when considering an agent or technique for intraoperative fluorescence imaging. The surgeon needs to be able to rely on fluorescence to truly highlight tumor and not normal, functionally intact, or edematous brain. However, defining or measuring selectivity of an agent for delineating tumor is difficult and wrought with numerous biases and error sources. For the sake of this article, it should be understood that measures of diagnostic accuracy (sensitivity, specificity) are not defined for intraoperative fluorescence imaging, depend very strongly on where and how many biopsies are collected, by which criteria a sample is being considered as having positive fluorescence, and many other factors. Thus, any reports on diagnostic accuracy regarding a fluorescing agent used for fluorescence-guided resections should be critically scrutinized.
Time-dependency of signal selectivity and signal strength
With the exception of autofluorescence, all fluorophores theoretically have a time dependency of their signal ( Fig. 1 ). For fluorophores injected intravenously without specific affinity to tumor cells, concentrations of fluorophore are at first high in plasma and thus in all perfused tissues, thereafter reaching the tumor via the broken-down blood-brain barrier. From there, extravasated fluorochrome more or less quickly dissipates with edema from the tumor, depending on the half-life in plasma. If these fluorochromes have specific affinity to tumor cells, for example, when coupled to a selective antibody, unbound fluorochrome will be washed out and bound fluorochrome will remain within the tumors. Metabolically activated fluorochromes will require a certain period of time to accumulate and will disappear with time. All fluorochromes are more or less subject to photobleaching, a process by which excitation light leads to degradation of fluorochrome with fading away of fluorochrome intensity.
Visibility of the fluorochrome to the surgeon
Not all fluorochromes are perceivable by the human eye. Protoporphyrin IX (PPIX) is clearly distinguished by its intense red fluorescence at 635 nm. Fluorescein is easily discernible with its yellow-green fluorescence due to a fluorescence maximum at 525 nm. ICG, an infrared dye first introduced for intraoperative fluorescence angiography by Raabe and colleagues, provides no fluorescence perceivable to the eye, with an absorption peak at 780 nm and an emission peak at 830 nm.
This article focuses mainly on ALA and fluorescein because these are the fluorochromes that are either approved or are being widely used in the clinical setting.
5-Aminolevulinic acid
ALA is paradigmatic as an agent inducing fluorescence in brain tumors. After being introduced in a first clinical study in 1998, ALA was the first to enter structured clinical development, culminating in a large-scale, state-of-the-art prospectively randomized clinical trial, which resulted in regulatory approval throughout Europe and many countries of the world. Although its use in the United States is still restricted to clinical trials of an investigational new drug, a new drug application was filed in December 2016 with approval expected in 2017. In the meantime, a clinical trial providing further supporting data has recently commenced in the United States (A Multicenter Study of 5-Aminolevulinic Acid to Enhance Visualization of Malignant Tumor in Patients with Newly Diagnosed or Recurrent Malignant Gliomas: A Safety, Histopathology, and Correlative Biomarker Study, principal investigator: Costas Hadjipanayis, Mount Sinai, New York).
Biochemical background and safety
ALA is a body’s own metabolite in the biosynthesis pathway. For reasons not completely unraveled, gliomas selectively take up ALA and convert ALA into PPIX via enzymes of this pathway. Selectivity of ALA-induced PPIX has been demonstrated to be high in many studies, although normal brain tissue has not been observed to accumulate PPIX in response to ALA exposure. All major modern surgical microscopes offer adjuncts with the ability of visualizing PPIX. Visualizing PPIX fluorescence requires filtered xenon light to give blue-violet light with a wave-length of 375 to 440 nm and an emission filter, allowing visualization of red fluorescence, which has a peak at 635 and 704 nm. The filters are also constructed to allow some of the excitation light and green autofluorescence emitted from the tissue to pass, thus enabling background discrimination and permitting extended parts of the surgery to be performed in the fluorescence mode.
ALA is administered as oral solution at a dose of 20 mg/kg body weight. Due to the small size of this molecule, it is rapidly absorbed from the intestine and is cleared from plasma within 2 hours after administration. Peak fluorescence can be expected after about 6 to 8 hours, with fluorescence beginning to become visible after about 3 hours.
Toxicologic safety is high, as indisputably confirmed in a large randomized study and more recently in other prospective cohorts.
Transient elevations of liver enzymes have been noted 24 hours after application of ALA, rapidly returning to normal levels thereafter. Because porphyrins transiently accumulate in the skin, the skin is temporarily light sensitive for the first 24 hours after application. This requires patients to be maintained away from direct light after surgery. The authors suggest pulling window shades and turning off direct lighting in the room, although we find ambient light to be safe. To the authors’ knowledge, there have been no reports of porphyria-like symptoms after the administration of ALA in the context of brain tumor surgery.
5-Aminolevulinic acid in malignant gliomas
Most work so far has been presented regarding the use of ALA in malignant gliomas, foremost glioblastomas. In 1998, the senior author, Stummer and colleagues published the first results of a small cohort of subjects with malignant gliomas and corroborated it by an additional series of patients in the year 2000. Most of these subjects were glioblastoma subjects, as was the case in a randomized phase III study. In that study, subjects were randomized to be operated on using ALA for identifying tumor compared with normal white light resections. The rates of complete resections were doubled when surgeons had ALA-induced fluorescence for identifying tumor. Although the degree of resection can be assumed to drive prognosis, this study with its particular design detected a significant prolongation only of progression-free survival, whereas overall survival was only marginally influenced. However, prognosis was being driven by extent of resection and, in the ALA cohort, 35% of subjects were not treated by complete resections in order not to impair neurologic functions, whereas in the control group 35% of subjects also had complete resections of contrast-enhancing tumor. In this study, resection was driving survival rather than the administration of 5-ALA. Importantly, the study was able to rule out extended resections using fluorescence to be associated with significantly greater neurologic morbidity.
Since this study, 5-ALA has been approved for fluorescence-guided resections in many countries of the world and is commonly used for glioblastomas. The gross total resection rates of 65%, as reported in the randomized study, have frequently been cited as the limit of what can be achieved using this method. However, the subjects operated in this study were the first subjects of the individual surgeons at that time and modern intraoperative monitoring and mapping were not used. Today, several series have been published that show much higher resection rates, especially when mapping and monitoring are used, including series focusing only on subjects with tumors in regions of the brain traditionally deemed eloquent, that is, the central region or the language regions ( Table 2 ).
Study | Number of Subjects | Eloquence | Monitoring and Mapping | Intraoperative MRI | Study Type | Resection Rate (%) | Comment |
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Stummer et al, 2000 | 50 | Eloquent and noneloquent | No | No | Prospective monocentric cohort | 65 | — |
Stummer et al, 2006 | 135 | Eloquent and noneloquent | No | No | Prospective multicentric 2-arm randomized | 65 | — |
Della Puppa et al, 2013 | 25 | Eloquent only (motor, language) | Yes | No | Prospective monocentric cohort | 80 | — |
Coburger et al, 2015 | 33 | Eloquent and noneloquent | Yes | Yes | Prospective monocentric cohort, historical matched pair | 100 | Intraoperative MRI alone 82% |
Diez Valle et al, 2011 | 36 | Eloquent and noneloquent | Yes | No | Prospective monocentric cohort | 83.3 | — |
Schucht et al, 2014 | 67 | Eloquent only (motor) | yes | No | Prospective monocentric cohort | 76 | — |
Schucht et al, 2012 | 103 | Eloquent and noneloquent | Yes | No | Prospective monocentric cohort | 96 | — |