Neurosurg Rev DOI 10.1007/s10143-014-0546-6

REVIEW

Fluorescein-guided surgery for malignant gliomas: a review Francesco Acerbi & Claudio Cavallo & Morgan Broggi & Roberto Cordella & Elena Anghileri & Marica Eoli & Marco Schiariti & Giovanni Broggi & Paolo Ferroli

Received: 21 August 2013 / Revised: 8 January 2014 / Accepted: 26 January 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Fluorescein is widely used as a fluorescent tracer for many applications. Its capacity to accumulate in cerebral areas where there has been blood–brain barrier damage makes it particularly suitable as a dye for the intraoperative visualization of malignant gliomas (MGs). In this report, we describe the results of a comprehensive review on the use of fluorescein in the surgical treatment of MGs. A comprehensive literature search and review for English-written articles concerning the use of fluorescein in the resection of MGs has been conducted. The search was executed through a PubMed literature search using the following keywords: malignant gliomas, glioblastomas, high-grade gliomas, YELLOW 560, total removal, dedicated filter, neurosurgery, brain tumors, intracranial tumors, and confocal microscopy. The literature search resulted in the retrieval of 412 evidence-based articles. Of these, 17 were found to be strictly related to the resection of MG with the aid of fluorescein. In addition to these 17, we have included 2 articles derived from a personal database of the corresponding author (FA). The analysis of the articles reviewed revealed three major applications of fluorescein during surgery for MGs that was documented: Fluorescein-guided resection of MGs with white-light illumination, fluorescein-guided resection of MGs with a surgical microscope equipped with a dedicated filter for fluorescein, and confocal microscopy for intraoperative histopathological analysis on MGs. The systemic review conducted on the use of fluorescein in MGs explored the applications and the different modalities in which F. Acerbi (*) : C. Cavallo : M. Broggi : R. Cordella : M. Schiariti : G. Broggi : P. Ferroli Neurosurgical Department, Foundation IRCCS Istituto Neurologico Carlo Besta, Via Celoria 11, 20133 Milan, Italy e-mail: [email protected] E. Anghileri : M. Eoli Molecular Neuro-oncology, Foundation IRCCS Istituto Neurologico Carlo Besta, Milan, Italy

fluorescein has been used. The data we have gathered indicates that fluorescein-guided surgery is a safe, effective, and convenient technique to achieve a high rate of total removal in MGs. Further prospective comparative trials, however, are still necessary to prove the impact of fluorescein-guided surgery on both progression-free survival and overall survival. Keywords Malignant gliomas . Glioblastomas . Brain tumors . Fluorescein . YELLOW 560 . Total removal . Dedicated filter . Confocal microscopy

Introduction Malignant gliomas (MGs) represent the vast majority of malignant brain tumors. According to the current WHO grading system classification, MGs include grade III and IV lesions [24]. These are rapidly progressive and invasive tumors which have a very poor prognosis even when maximal therapy (surgery plus radiotherapy (RT) and chemotherapy (CHT)) is used [42]. Though the role of radical resection still remains controversial, there are some retrospective studies and one prospective study that suggest it to have a positive effect on survival of patients with MGs [16, 22, 25, 37]. However, the complete resection of the contrast-enhanced part of the lesion, the so-called gross total resection (GTR), has been reported to be feasible only in a low percentage of cases [22, 25], due to difficulties in recognition of the tumor tissue at the margin of resection [46]; this appears to be the case in spite of the introduction of many innovative technologies, such as neuronavigation [49], intraoperative MRI [15, 23, 29], and/or cranial ultrasound [10, 47]. The use of fluorophores in order to enhance and facilitate the discrimination between tumor and normal tissue has been

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reported in different surgical specialties, including neurosurgery [27]. 5-aminolevulenic acid (5-ALA), a natural precursor of hemoglobin that promotes the metabolic synthesis of fluorescent porphyrins in cancerous cells, has also been used in MGs, with the result of a significant increase in the extent of resection [4, 38–41] and a consequent improvement in progressionfree survival (PFS) at 6 months [39]. However, there are a number of factors that limit the widespread use of 5-ALA fluorescence as a guide for the resection of MGs. Included among these factors are the need to administer the drug orally 2.5 to 3.5 h before induction of anesthesia, the need to avoid direct exposure of patients to sunlight or strong room light for 24 h after using 5-ALA because of the risk of skin sensitization [46], and finally, the high costs of 5-ALA (around 900 € for each vial). Another possible fluorophore that can be used in neurosurgery is sodium fluorescein, the sodium salt of fluorescein. This biomarker is excited by a light wavelength ranging from 460 to 500 nm and emits fluorescent radiation in the wavelength ranging from 540 to 690 nm. It is usually available as a water-soluble dye that has been extensively used in the field of ophthalmology since 1961, particularly to perform retinal angiography, with very few side effects [20, 21, 30, 50]. The mechanism of action of fluorescein is different from that of 5-ALA. Fluorescein usually accumulates in specific cerebral areas as a result of leakage from a damaged blood–brain barrier (BBB). In the case of the MGs, the invasiveness of these lesions changes the morphology of the BBB and disrupts its continuity along brain vessels modifying their permeability. This allows fluorescein to become concentrated specifically at the tumor site, making the tumor tissue more clearly visible, particularly if a dedicated filter on the surgical microscope is available, with a possible increase in GTR rate [1] (Figs. 1 and 2). It should also be noted that fluorescein is inexpensive compared to the costs of 5-ALA (5 € for each vial) and that can be easily intravenously administered at the moment of anesthesia induction. Further support for fluorescein utilization comes from the ophthalmologic literature where its use has been reported with few side effects [20, 21, 30, 50]. Our intent was to conduct a comprehensive review on the application of fluorescein during surgery for MGs.

Methods A comprehensive review of the most relevant published articles in the English language since 1947 on resection of MGs by the aid of fluorescein has been completed using a PubMed search with the following keywords: fluorescein,

Fig. 1 Preoperative and postoperative MRI study and intraoperative images of a case of left frontal glioblastoma operated on with fluorescein-guided technique by the use of YELLOW 560 filter. a Preoperative MR (T1 with gadolinium), showing a left frontal partially cystic tumor, compatible with a diagnosis of malignant gliomas. b Postoperative MR (T1 with gadolinium), showing the complete resection of the tumor. c Dural exposure after craniotomy with white-light illumination. d Dural exposure after YELLOW 560 filter activation, showing the yellow-green discoloration due to the fact that in dural tissue no blood-brain barrier is present. e After dural opening, brain parenchyma is exposed, without clear demonstration of the location of the tumor. f After YELLOW 560 filter activation, there is a clear delineation of the tumor area (white arrow) with respect to the peritumoral brain. g At the end of the removal, no residual tumor tissue is evident. h The absence of residual tumor is confirmed by the activation of YELLOW 560, showing no fluorescent areas. Histological diagnosis showed a glioblastoma. The patient suffered from a slight worsening of language disturbances in the early postoperative period

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Results This systemic evidence-based literature search resulted in 412 articles. Of these, 17 were found to be strictly related to the resection of MGs by the aid of fluorescein. In addition, 2 articles coming from a personal database of the corresponding author (FA) have been included. The analysis of the articles selected in our literature search revealed three major applications of fluorescein during surgery for MGs. Fluorescein-guided resection of MG with white-light illumination (Table 1)

Fig. 2 Preoperative MRI and intraoperative images of a case of left temporal glioblastoma operated on with fluorescein-guided technique by the use of YELLOW 560 filter. a Preoperative MR (T1 with gadolinium) showing a huge mass of enhancing tumor, compatible with a diagnosis of malignant glioma. b During removal with ultrasonic aspiration with white-light illumination, it was difficult to clearly distinguish tumor tissue (white arrow) from peritumoral areas. However, the activation of YELLOW 560 Filter (c) allowed for the identification of the fluorescent tumor (white arrow). d, e postoperative MR T1 images without (d) gadolinium showed the surgical cavity with hyperintensity due to hemoglobin products, while T1 with gadolinium (e) confirmed the absence of contrast enhancement, thus showing GTR of the tumor. Histological diagnosis showed a glioblastoma. The patient suffered from a slight worsening of language disturbances in the early postoperative period

malignant gliomas, glioblastomas, high-grade gliomas, YELLOW 560, total removal, dedicated filter, neurosurgery, brain tumors, intracranial tumors, and confocal microscopy. Our review has been restricted to the application of fluorescein in MG, and we have not reviewed the literature of that dye in the treatment of histological tumor types different from MGs nor have we considered the literature concerning other dyes beside fluorescein. We have also chosen to exclude reviews that discuss the more general question of fluorescence-guided removal of MGs. Few papers of the personal database of the corresponding author (FA) were included in the review.

The first reported application on the use of fluorescein during surgery of MGs was described by Moore in 1947 [26, 27]. In this study, 46 patients received 1 g of fluorescein intravenously immediately before or during ventriculography, in order to confirm the location of subcortical tumors. The majority of tumors detected by needle biopsy were malignant astrocytomas. The material sampled suggested the presence of tumors sometimes by direct white-light visualization but usually by ultraviolet light directly in the operating room. The biopsy was then verified by histological examination, with a correct diagnosis assured by fluorescein visualization in 96 % of cases. Similar applications were reported in subsequent series [2, 43]. The accuracy of fluorescein to identify tumor tissue was systematically studied by Murray, by performing a total of 186 biopsies in 23 patients with brain malignancies. In that study, he obtained a sensitivity of 96 % and specificity of 81 % [28]. The use of fluorescein to improve the percentage of resection of MGs has only been explored in recent years. Some studies were performed without the aid of a specific illumination light and filter to allow for a better discrimination of fluorescein fluorescence [3, 14, 36]. Shinoda et al. in 2003 reported the first retrospective analysis on a series of 32 patients operated on with the aid of fluorescein, but without a special filter adapted to the surgical microscope [36]. The operation was therefore completed under white-light illumination and with a standard surgical microscope. In this case, a high dose of fluorescein was intravenously administered (20 mg/Kg) in order to detect fluorescence in the surgical field, at the time of dural opening. For this reason, immediately after intravenous injection, the normal cortex, vessels, dura mater, and tumor were stained yellowish, but after approximately 5 min only, the tumor retained the yellow dye. The yellow appearance was different inside the tumor, with the center more deeply stained and the periphery faintly stained. However, even the periphery appeared clearly discernable from the negative peritumoral area. This

Neurosurg Rev Table 1 Fluorescein application during surgery for malignant gliomas with white-light illumination Fluorescein’s application

Authors

Year No. of patients

Amount of fluorescein

Time of fluorescein injection

Localization of brain tumor Localization of brain tumor

Moore [27] Moore et al. [26]

1947 12 1948 46

1,000 mg 1,000 mg

1951 –

–

Not specified Not investigated Before or upon the completion Not investigated of ventriculography (approximately 2 h before surgery) – –

1951 Not specified

1,000 mg

Not specified

Not investigated

Identification of brain tumor Murray [28]

1,000 mg

Not specified

Not investigated

Resection of MG Resection of MG Resection of MG

20 mg/Kg Before dural opening 20 mg/kg Before dural opening 15–20 mg/Kg Before dural opening

Localization of brain tumor Belcher et al. [2] (theoretical evaluation of the physical limitations of the method) Localization of brain tumor Svien et al. [43]

1982 23 (14 MGs) (for 186 biopsies) Shinoda et al. [36] 2003 32 Koc et al. [14] 2008 47 Chen et al. [3] 2012 22

difference in fluorescence seemed to be correlated to the histological characteristics of the tumors. In fact, in four of the cases analyzed by histology, the authors found glioblastoma (GBM) tissue in the center deeply stained area, while less dense tumor cells and scattered endothelial proliferation in the faintly stained area. Based on postoperative MRI, interestingly enough, the percentage of GTR was almost 85 %, and it was significantly better than the percentage of 30 % obtained by the same authors in a historical control group operated without fluorescein. However, no statistically significant difference in survival could be found between these groups. Similar results were reported by Koc et al. [14], who described the result of a prospective analysis on two nonrandomized groups, one of which operated by the aid of fluorescein (20 ml/Kg at the time of dural opening) without a specific filter on the microscope (group 1), and one without fluorescein administration (group 2). The percentage of GTR based on postoperative MRI was 83 % in group 1 and 55 % in group 2. Both groups were submitted only to RT, without concomitant or adjuvant CHT. Overall survival was therefore poor, with 44 weeks in group 1 and 42 weeks in group 2, without a significant difference. A similar trial was performed by Chen et al. [3], reporting a significant increase in GTR in patients who undergone fluorescein-guided surgery for MGs. A total of 22 patients were enrolled in this study, divided into the study group (n=10) which included patients that received intravenous fluorescein injection (15–20 mg/Kg at the time of dural opening) and the control group (n=12) without fluorescein administration. GTR rate was 80 % in the study group and 33.3 % in the control group. No complications were detected by Chen et al. [3] in a study where preoperative allergy test were performed in order to avoid any possible allergic reactions. Persistent yellow staining of skin, mucosa, and urine was the only adverse event observed by Shinoda

GTR rate (%)

84.4 83 80

et al. [36] in the next 24 h after operation. Even though a low complication rate has been described in the ophthalmologic field [20, 21, 30, 50], some side effects can occur after highdose fluorescein administration. Few anaphylactic reactions have been reported with severe bradycardia and hypotension [5, 45]. Fluorescein-guided resection of MG with a surgical microscope equipped with a dedicated filter for fluorescein (Table 2)

Due to the specific characteristics of fluorescein, with a maximum absorption at 494 nm and maximum emission of 521 nm, some filters have been developed to help in the discrimination of fluorescence during surgical approaches for MGs removal. The first experiences were made with filters not directly integrated in the surgical microscope, but adapted to guarantee a visualization of fluorescein that was coherent with its absorption and emission wavelength peaks. Kabuto et al. in 1997 used two filters for the excitation and emission of fluorescein that could be manually fitted to and removed from a surgical microscope during operation for MGs in five cases [12]. These authors, injecting a high dose of fluorescein (1 g i.v.), reported a clear visualization of the tumor under the filter, but they also found a faint yellow color under white-light illumination. Observation during tumor removal could be changed to ordinary white-light illumination by removing the filters, in order to perform surgical maneuvers requiring a better visualization of the structures in and around the tumors, such as vessels. A similar combination of high-dose i.v. fluorescein (20 mg/ Kg upon dural opening) and excitation and barrier filters

75

100 Not investigated

100 83.3 95

Not investigated

5 mg/kg 20 (ongoing trial) Acerbi et al. [1] YELLOW 560 filter integrated in the Pentero microscope (Carl Zeiss)

2013

20 mg/kg 3–4 mg/kg Okuda et al. [31] Schebesch et al. [34] Inserted in the operating microscope (OME-9000, Olympus) YELLOW 560 filter integrated in the Pentero microscope (Carl Zeiss)

2012 2013

Kuroiwa et al. [19] Kuroiwa et al. [18] Kuroiwa et al. [17] Integrated in the microscope (OPMI, Carl Zeiss) Integrated in the microscope (OPMI, Carl Zeiss) Integrated in the microscope (OPMI, Carl Zeiss)

1998 1999 1999

10 (8 with tentative GTR) 30 20 (3 combined with frameless stereotactic system) 10 (5 with tentative GTR) 35

8 mg/kg 8 mg/Kg 8 mg/Kg

Before tumor resection (not further specified) Upon dural opening Upon dural opening Before incision of the dura mater Upon dural opening After bone flap removal, prior to durotomy After anesthesia induction, before skin incision 1,000 mg 5 (3 MGs) Kabuto et al. [12] Manually fitted and removed

1997

No. of patients Year Authors Dedicated filter

Table 2 Fluorescein application during surgery for malignant gliomas with specific filters in the surgical microscope

Amount of fluorescein

Time of fluorescein injection

GTR rate (%)

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inserted in the operating microscope (OME-9000, Olympus) was proposed more recently for glioblastoma (GBM) removal, by Okuda et al. [31]. Ten consecutive patients were included in this trial, and no side effects were described. Depending on the resection requirements, the high fluorescein dose allowed surgical maneuvers to be performed both in fluorescence mode and under normal white xenon-light illumination. According to these authors, this seemed to improve the reliability of surgical removal of GBM. In all the five cases studied with an intended GTR, this was confirmed postoperatively. Kuroiwa et al. described for the first time a technique in which the fluorescence filter was directly integrated in the microscope (Zeiss OPMI) [19]. In this study as well, visualization could be switched during operation from filter to whitelight illumination. In addition, due to specificity of the filter for absorption and emission wavelength of fluorescein, these authors were the first to use a reduced dose of i.v. fluorescein, specifically 8 mg/kg, which was injected upon dural opening. As reported by Shinoda, without filter visualization [36], they documented immediately after injection a fluorescence staining in the arteries, veins of the surface, brain parenchyma, and tumor. But 5 min after injection, the brain surface fluorescence lessened, and tumor removal started 20 min later. Ten patients were included in this study. In 8 cases, a complete tumor removal could be obtained. The same group [18] studied a larger series of 30 patients using the same technique, and in 5 cases, they compared the intraoperative finding with standard histological analysis. Fluorescein-positive areas revealed abnormal tumor vessels and dense tumor cells, while fluorescein-negative areas showed only scant tumor cells infiltration and no abnormal vessel. In a different paper, the same group confirmed the usefulness of fluorescein during MGs removals, even when associated with frameless stereotactic technique for deep contrast-enhancing brain tumors [17]. Since 2012, a dedicated module for fluorescein filter (YELLOW 560) has been specifically designed by the Carl Zeiss Company (Germany) for excitation in the wavelength range from 460 to 500 nm and for observation in the wavelength range from 540 to 690 nm, and directly integrated in the Pentero surgical microscope. The YELLOW 560 module improved visualization of the pathological and normal anatomy in the fluorescent mode [1]. In fact, once the YELLOW 560 mode had been activated and the filters moved into the microscope’s light path, specifically defined amounts of blue and red light were mixed for generating a white-light impression of the non-fluorescent tissue. This allowed for a delineation of both the fluorescent signal and the non-fluorescent tissue in a more natural color. In this way, it was possible to protect the normal structures during surgical maneuvers under fluorescence visualization [1]. Furthermore, due to the high specificity for fluorescein of this filter, the fluorescein dose could be further reduced in clinical application [1, 34].

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In 2013, two papers concerning the use of Yellow 560 filters for surgery of MGs have been published [1, 34]. Schebesch et al. [34] conducted a retrospective analysis on 35 patients, including 22 with MGs (12 initial glioblastoma, 5 recurrent glioblastoma, and 5 grade III gliomas), operated on from May to August 2012. A total of 2 ml (200 mg corresponding to approximately 3–4 mg/kg) of fluorescein was intravenously administered after bone flap removal prior to durotomy. Most of their results came only from a qualitative analysis, and there was no definitive data presented regarding the extent of resection in MGs. In addition, no histological studies on fluorescent and non-fluorescent tissue were performed. In this study, there were no adverse reactions registered. With this lowdose of fluorescein, only the urine became colored yellow for 6 h postoperatively with no sclera or skin discoloration experienced by the patients. Our group was the first to propose a phase II prospective trial on fluorescein-guided resection of MG with a dedicated filter on the surgical microscope (FLUOGLIO study) [1]. This study aimed to evaluate safety and to obtain initial information about the efficacy of fluorescein-guided removal of MGs. The first patient was enrolled in September 2011, and since then, 20 patients have been included. In the first year of the study, when the YELLOW 560 module was not available yet, we used the BLUE 400 module that was previously developed for 5-ALA surgery. Due to its wide range of emission and excitation, this filter also allowed for good visualization of the light emitted by fluorescein in the area where there was BBB disruption. In particular, there was good discrimination between the fluorescent area, which appeared yellowish green, and the non-fluorescent tissue, which was colored dark blue [1]. The only problem that remained for us was that we still had to use an intermediate dose of 10 mg/Kg to clearly visualize the fluorescent tissue. This dose was established in a preliminary study during surgery for MGs (Acerbi, unpublished data). Since 2012, all the patients included in the study have been operated on by the use of the YELLOW 560 module on the Pentero microscope (Carl Zeiss, Germany). With this filter, the total fluorescein dose was reduced to 5 mg/Kg. In addition, contrarily to other experiences, fluorescein was intravenously injected after the anesthesia induction and before skin incision. This was decided in order to avoid manipulation of the cerebral tissue before fluorescein accumulation in the areas of BBB damage. Our preliminary report on 12 patients with GBM with a median preoperative volume of 33.15 cm3 [1] showed that no side effect due to fluorescein administration could be registered in the trial and that GTR was achieved in 75 % of cases. In addition, a measure of accuracy of fluorescein in identification of tumor tissue showed a sensitivity of 91 % and specificity of 100 %.

Confocal microscopy for intraoperative histopathological analysis on MG Confocal microscopy is an innovative technology that has been developed in the last decade. Its introduction represents a major breakthrough in the conventional histopathological analysis for selected tissue biopsy. Confocal microscopy has been integrated in different medical specialties, since its initial application in gastroenterology, where it was used to evaluate endoscopically inapparent Barrett’s esophagus [6, 13] or colorectal cancer [11]. It has also been used for endomicroscopic diagnosis of cervical intraepithelial neoplasia [44] or uroepithelial cancer [48]. Its application in the neurosurgical field followed in turn. But in order to do this, a specific tool needed to be developed [7, 32, 33]. The essential element of this neurosurgical tool was a rigid probe containing a miniaturized scanner that allowed an operator-dependent specific depth-visualization that was usually in the range of 0–250 μm. This technical tool was handheld and sterilizable, and perfectly designed for neurosurgery. The scanner was connected to an external monitor in order to visualize the tissue against which the probe was directed, which was then sent to a personal computer unit. This technique allowed an in vivo visualization of the histopathological features and cytoarchitecture of the analyzed samples, giving a significant aid in the intraoperative tumor identification [7]. Furthermore, this approach seemed to overcome the limitations presented by the traditional intraoperative frozensection evaluation that can be subject to mechanical or chemical alteration or can present artifacts [7]. In the studies performed by Eschbacher et al. and Sanai et al. from the Barrow Neurological Institute [7, 32], the clinical protocol included 25 mg of i.v. fluorescein sodium in order to increase the qualitative difference of the contrast-enhanced tumor margin. According to this study, the imaging process was initiated 5 min after fluorescent tracer administration. Once the different surgical sites were studied and the images acquired, biopsy samples were taken in order to perform the traditional histological analysis. In the blinded study that was performed [7], a neuropathologist interpreted the confocal images and the biopsy samples. In 28 images, an accuracy of 92.9 % was reported. As these data suggest, this relevant device was less time-consuming, and it had a comparable sensitivity to the traditional frozen-section analysis.

Discussion Our review on articles published in English since 1947 has considered 19 articles related to resection of MGs by the aid of fluorescein. Seventeen of these articles were related to the application of fluorescein during MGs surgery, with the purpose of improved tumor tissue visualization. Some of these studies suggested the utilization of high-dose intravenous

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fluorescein administration for biopsies of MG, in order to confirm localization of the tumor (in the early published series before neuroimaging) and, more recently, to obtain intraoperative correct specimen samples [2, 26–28, 43]. The natural evolution of the technique was related to the application of fluorescein during MG removal in order to obtain an increase in extent of resection. In fact, though there is still debate in the neuro-oncological literature, there is some recent evidence to suggest a possible prognostic effect of total resection on survival of patients with MGs [16, 22, 25, 37]. However, GTR of the tumor, i.e., removal of the whole enhancing part of the MRI, was considered to be feasible in a low percentage of case [22, 25], due to the difficulties in recognizing the pathological tissue at the tumor margins [46]. The retrospective series published by McGirt et al. in 2009 [25], in which they considered patients operated on in the period 1996–2007 that excluded locations precluding a complete removal, a GTR was feasible only in 39 % of the cases at the first diagnosis. These results were similar to those obtained from the control group of patients of the 5-ALA study, which had been operated on without the aid of fluorescent visualization. In this latter group, a complete resection was reported to be feasible only in 36 % of the cases [39]. A number of technical tools, such as neuronavigation [49], intraoperative magnetic resonance imaging (iMRI) [15, 29], and ultrasound [47] were developed to obtain better surgical results, but only a randomized trial on iMRI was performed demonstrating a class I evidence of its application on extent of resection [35]. In addition, the use of fluorophore to enhance tumor visualization has been demonstrated to be useful during surgery for

MGs (Table 3). In particular, the utilization of 5-ALA has been shown to increase the rate of complete resection and the 6 months PFS in a randomized trial [39]. From the analysis of the literature, fluorescein injection seemed to be a good method to obtain a high rate of GTR during surgery for MGs. Percentage of resection in the analyzed series varied from 75 to 100 %. In some papers, a significant increase of total resection was obtained if compared with cases that had been operated on without fluorescein use [3, 14, 36]. In addition, even if the mechanism of action is not related to a specific uptake of fluorescein by tumoral cells (as for 5-ALA), thus allowing for the possibility of false-positive cases (metastases, surgical trauma, etc.), different studies performed with and without filters on the surgical microscope showed a high level of accuracy in tumor identification [1, 18, 19, 28]. It should be mentioned also that the low cost of fluorescein (around 5 € for each vial), compared to 5-ALA, makes this alternative much more attractive. However, it needs to be noted that a proper phase III trial has not been published which is crucial to provide the kind of definitive data concerning any possible increase in PFS as well as the survival of patients operated with fluorescein-guided technique. It is also important to recognize, additionally, that even with a fluorescein-guided technique and under specific filter visualization, a complete removal of favorable MGs is not always possible. As for other types of fluorescence tools currently been used in neurosurgical applications [8, 9] and as it has been suggested for 5-ALA [46], it is possible to enhance the discrimination of the pathological tissue with fluorescein only if it is properly exposed during the surgical approach. In two of the cases included in

Table 3 Comparison between fluorescein and 5-aminolevulenic acid for surgery of malignant gliomas 5-Aminolevulinic acid (5-ALA)

Fluorescein

Dosage Time and mode of injection

Drug: precursor of hemoglobin Selectively elicits the synthesis of fluorescent porphyrin IX in tumoral cells 20 mg/Kg 2–3 h before surgery, orally administered

Dye: sodium salt of fluorescein Passage throughout the damaged blood-brain barrier (BBB) 3–10 mg/Kg At the time of surgery, intravenously administered

Fluorescence visualization

Specific filter for visualization: needed (ZEISS-LEICA)

Specific filter for visualization: needed (unless high dosage) (ZEISS) Filter characteristics: good discrimination of fluorescent tissue; visualization of peritumoral area in more natural colors 75–100 %a No definitive data available No definitive data available Around 5 € for each vial

Type of substance Mechanism of action

Filter characteristics: good discrimination of fluorescent tissue; poor visualization of peritumoral area

Effect on GTR rate Effect on PFS Effect on overall survival Costs

65 vs 36 %b 6-PFS 41 vs 21.1 %b No definitive data available Around 900 € for each vial

a

Data derived from the present review, including only case series, retrospective studies, and prospective non-randomized controlled studies

b

Data from a randomized controlled multicenter study (against white-light illumination)

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the FLUOGLIO study from our group [1], a small remnant was left due to its location around the corner of visualization during the approach. In addition, sometimes, in tumor located in close proximity to eloquent areas, a portion of fluorescent tissue could be left at the tumor margin if cortical or subcortical brain mapping shows positive responses. The use of specific filters on the surgical microscope seemed to provide a better delineation of the tumoral tissue. Unlike the filter used for 5-ALA-guided removal, however, the YELLOW 560 module on the Pentero microscope assured the visualization at the same time of the fluorescence areas and the peritumoral brain parenchyma and vessels in more natural colors thus allowing performing most of the procedure with the fluorescence module activated. In addition, it permitted a high-definition motion to be captured for video recording and subsequent analysis. Furthermore, the use of specific filters enabled the reduction of the total dose of fluorescein injected [1, 19, 34] with a possible reduction of side effects. In fact, even if adverse events after fluorescein injection have been rarely reported in ophthalmological and neurosurgical literature [5, 20, 21, 30, 45, 50], no side effects were registered with the use of a low-dose fluorescein in more recent papers. It should also be noticed that in neurosurgical application, side effects are probably even less frequent than in ophthalmological cases due to the fact that fluorescein administration was performed with the patient intubated under general anesthesia (Table 3). Confocal microscopy is a new interesting application derived by the use of fluorescein to enhance tumor discrimination [7, 32, 33]. Clinical use during surgical approaches for MGs suggested the high level of accuracy of confocal microscopy with high-dose fluorescein in identification of pathological tissue at the tumor margin [7]. In addition, this technique appeared to be less time-consuming though with a comparable sensitivity to the traditional frozen-section analysis. Finally, we could speculate that a combination of fluorescein-guided resection of MGs by the use of a dedicated filter on the surgical microscope and confocal microscopy to confirm the absence of cancerous residual cells at the tumor margin represents an exceptional method to extend the surgical removal to a cellular level.

Conclusions The presented systemic review has explored the applications and different modalities of fluorescein utilization. Emerging data seem to demonstrate that fluorescein-guided surgery is a safe, effective, and convenient technique in achieving a high rate of GTR. Further prospective comparative trials, however, do need to be performed to definitively prove its effect on both PFS and overall survival.

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Comments Peter Nakaji, Phoenix, USA In this review, Acerbi et al. from Paolo Ferroli’s group at the Besta in Milan describe the literature and their personal experience using fluorescein as a fluorophore to guide the resection of malignant gliomas. This technique involves the preoperative injection of intravenous fluorescein

Neurosurg Rev and subsequent visualization under a microscope designed to detect fluorescence in the 560 nm range. The brain lights up in yellow under white-light visualization and, generally, the tumor lights up even more, although it varies. This is where things get interesting. Fluorescein is a non-specific fluorophore whose concentration in the tumor relies upon the increased vascular permeability of the tumor compared to normal brain. The advantage of fluorescein compared to metabolized fluorophores such as 5-ALA is that it can be seen while still in the unaugmented visual range, allowing the surgeon to operate and still see the whole surgical field. The tumor generally does appear even brighter yellow. The hope of all of us who are working with fluorescein is that it can be used to guide resection. The underlying premise of cytoreductive surgery is that a lower volume of tumor correlates with better control of the tumor. While there is evidence for this, there is both nuanced debate on this subject and the concern that increased resection in some locations will entail greater risk of deficit, with attendant impact on both survival and functional outcome. These challenges face not only fluoresceinguided resection of malignant gliomas but also all resection guided by intraoperative adjuncts, whether they be visual indicators such as 5-ALA, image guidance, ultrasound, asleep mapping, awake surgery, or endomicroscopes. The principal advantage gained in the use of these agents is if they improve our resection of the marginal tissue whose appearance blends with normal brain. Fluorescein is fairly non-specific, but with experience, there is mounting reason to think that it can help us make this particular distinction. Consideration of the merits of fluorescein immediately invites a deeper comparison with 5-ALA. 5-ALA is a prodrug which is used in the heme synthesis pathway, and in most gliomas, undergoes conversion into protoporphyrin IX, which fluoresces in the 400-nm (ultraviolet) range. It is more specific in the sense that tumor cells preferentially metabolize it, and therefore, it is concentrated in the target tissue. In theory, high specificity of a dye for the tumor is advantageous, but as tumor cells are always found beyond the visible margin, macrofluorescence alone may drive under-resection. At the same time, careful removal of only macrofluorescent tumor also may not be enough to prevent deficits, if the normal tissue is so closely entwined with the tumor that its manipulation alone mandates tampering with eloquent structures. The debate continues on. Perhaps the best role for these agents is in limiting the unintentional leaving of small amounts of residual tumor in non-eloquent areas. In practical terms, fluorescein has advantages that are considerable for intraoperative use. Fluorescein is a molecule with a long history of safe human administration and a low cost around the world. As the authors recognize, questions about fluorescein remain. The exact dose and timing that is optimal remain to be worked out. The normal brain does fluoresce somewhat; the threshold for distinguishing tumor from brain from necrosis has yet to be firmly established and to be rigorously confirmed through correlative histology. In the future, surgery may benefit from fluorophores that are highly specific to high-grade glioma—and indeed to any other type of tumor—helping surgeons to identify more accurately what is tumor and what is not, while other technologies tell us whether that tissue can be removed safely. In the meantime, this kind of intermediate technology has advantages that bear continued exploration as the authors and others are doing. Andreas Raabe, Bern, Switzerland This interesting article reviews the use of sodium fluorescein as a dye to visualize tumor tissue during surgery of malignant gliomas. These invasive and often poorly delineated tumors may be “gross total resection (GTR)” eligible, or more precisely, “complete resection of enhancing tumor (CRET)” eligible in many cases, but GTR or CRET are achieved in a disappointingly low percentage of patients only. Depending on the selection of patients, this percentage ranges from 20 to 60 % even when neuronavigation or intraoperative ultrasound is used. The only “resection enhancing” technologies that are proven to detect concealed tumor tissue are intraoperative MRI and fluorescence imaging, both of which were shown to increase the CRET rate in eligible glioblastoma patients to 65 to 95 %.

Sodium fluorescein fluorescence is a promising technology for glioblastoma surgery. The integration of a specific yellow light filter into the surgical microscope makes it a straightforward intraoperative technique. The experience and images suggest that this method adds to the surgical artillery that improves the extent of resection. However, as the authors emphasize, hard data are still missing, and a randomized controlled trial comparing white-light and sodium fluorescein is needed to quantify the effect on glioblastoma surgery. In my view, different methods have different advantages, and the surgeon should have all of them in his or her toolbox to decide what is best for any given situation and patient. When 5-ALA and sodium fluorescein are compared in this review, we should keep in mind that most data about the latter technique are from single-center retrospective studies. Although I agree with the authors that this technology has the potential to increase the extent of resection, the numbers in Table 3 are somewhat biased in favor of the authors’ method and remain to be proven. How much tumor tissue is included in the sodium fluorescein fluorescence remains unclear. While 5-ALA is metabolized within the tumor cell, sodium fluorescein appearance in the glioma tissue depends on blood–brain-barrier (BBB) leakage. Taking contrast-enhanced MRI as a marker for BBB damage, we expect that this is the volume stained with extracellular sodium fluorescein. In contrast, intracellularly metabolized 5-ALA fluorescence is found beyond the BBB-damaged tumor volume, and the tissue volume resected with 5-ALA is indeed larger than the T1gadolinium-enhancing tumor [1]. A methodological advantage of sodium fluorescein over 5-ALA is unlikely; however, the low price for this drug may be a deciding factor in many countries. References 1. Schucht P, Knittel S, Slotboom J, Seidel K, Murek M, Jilch A, Raabe A, Beck J (2014) 5-ALA complete resections go beyond MR contrast enhancement: shift corrected volumetric analysis of the extent of resection in surgery for glioblastoma. Acta Neurochir (Wien) 156(2):305–12 Walter Stummer, Münster, Germany I gladly grasp the opportunity to write a comment to the review of Dr. Acerbi and co-workers which is being published in Neurosurgical Review on the use of fluorescein sodium for enhancing surgery of malignant gliomas. Fluorescein sodium has been around a while and was first used by Moore et al. 1948 [1] for finding gliomas when CT and MRI was still science fiction at best. Even in those early reports, the authors were aware of the lack of selectivity of fluorescein for these malignant tumors, noting that a “brilliant yellow-green fluorescence characterizes tumor tissue whereas the normal brain, which retains appreciably less dye, appears white. Edematous tissue surrounding the tumor does fluoresce, but to a lesser degree so that it can be readily separated both from normal brain and the tumor itself.” The senior neurosurgeons reading this comment will remember that after the reports by Moore and his colleagues, fluorescein has not found its way into neurosurgery, but was initially abandoned. A second round with fluorescein occurred in the late 1990s by Kuoriwa and co-workers from Japan, and three publications can be found by this group, the last in 1999 [2, 3, 4]. This group has also stopped using fluorescein sodium, switching to ALA. To date, they have published a considerable number of articles on malignant gliomas and ALA, the last in 2013 [5, 6, 7, 8, 9, 10, 11, 12]. So why did surgeons not continue using fluorescein sodium for brain tumor surgery? The answer is simple. Fluorescein sodium is hardly specific for tumor because it is a blood-borne dye. Wherever there is blood, there will be fluorescein. Is this a good basis for the surgeon to rely upon for intraoperative tumor identification? So today, we are seeing a second renaissance of fluorescein sodium, as reflected by the present review. But what is new? It is certainly not the biology of the dye. Fluorescein sodium is still

Neurosurg Rev located in the blood and leaks into the tumor through the broken down blood–brain barrier. What is new is that a novel filter system is available, the Yellow 560 filter, which is now being provided to the community by a microscope company. In contrast to older systems, which visualized yellow fluorescein fluorescence on a black background, this filter system retains background tissue information. What the surgeon sees is (almost) the normal brain and the superimposed fluorescein signal. Please note that this system is superb for vascular surgery, and in this particular context, a real advance. With the new system, the surgeon does not have to rely on the playback on an external video screen with the established indocyanine green (ICG), because ICG fluorescence is invisible to the human eye. As opposed to ICG, fluorescein fluorescence in vessels is clearly visible to the human eye. Thus, the surgeon can directly inspect and manipulate the vessels, which is a real advantage, and extravasated fluorescein is not of importance. But is fluorescein sodium with this filter also reliable for tumor surgery? As a matter of fact, the image initially appears nice, with the brain being beautifully visible in the background, and the fluorescein fluorescence appearing somewhat selective in the foreground. But be careful; this image is deceptive, since much of the weaker and aberrant fluorescein signal in vessels, edema, and blood in the cavity is now obscured by the additional, bright tissue background information, particularly in the late stages of surgery where discrimination becomes especially important. Normally, with fluorescence, the background is maintained dark to not lose the information that fluorescence conveys. In the context of the YELLOW 560 filter, the surgeon believes he is receiving selective information, where in fact, he is only seeing peak fluorescence. The filter system simply does not change the biology of the dye in the blood. The reader may wish to see an example of the use of fluorescein for glioma surgery in a video posted on abcNEWS and freely available (http://abcnews.go.com/Health/makingbrain-tumors-glow-saves-lives/story?id=17076243). Please note the fluorescein, which is in all vessels, is distributed weakly throughout the normal brain and is visible in the dura, the CSF, especially at the resection edge due to tissue injury, and even in the remote brain locations obviously injured during the course of resection. The reader of the review by Dr. Acerbi and co-workers should give this aspect some thought, as he should critically give thought to the authors’ assertion that fluorescein is so much cheaper than ALA, which is true. However, fluorescein has not been tested in expensive randomized GCP conform clinical studies for safety and efficacy in many hundreds of patients, as was ALA, nor has it been approved for brain tumor surgery, as is ALA. In all studies, so far fluorescein has been used off-label. This in itself bears a number of implications. The authors do not mention this important aspect, and the uncritical user should be well aware of this, especially if his patients experience ill side effects of fluorescein, which have been reported [13]. In the end, the authors themselves acknowledge the necessity of controlled clinical studies to elucidate safety and value of fluorescein in conjunction with novel technology. The community is certainly looking forward to those studies in the ongoing effort to increase the quality of our surgical care while keeping economical restraints in mind. References 1. Moore GE, Peyton WT, French LA, Walker, WW. The clinical use of fluorescein in neurosurgery; the localization of brain tumors. J Neurosurg. 1948 Jul;5(4):392–8. PubMed PMID: 18872412.

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