See the corresponding editorial in this issue, pp J Neurosurg 115: , 2011

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1 See the corresponding editorial in this issue, pp J Neurosurg 115: , 2011 Intraoperative confocal microscopy in the visualization of 5-aminolevulinic acid fluorescence in low-grade gliomas Clinical article Nader Sanai, M.D., Laura A. Snyder, M.D., Norissa J. Honea, M.S.N., Stephen W. Coons, M.D., Jennifer M. Eschbacher, M.D., Kris A. Smith, M.D., and Robert F. Spetzler, M.D. Barrow Brain Tumor Research Center, Barrow Neurological Institute, Phoenix, Arizona Object. Greater extent of resection (EOR) for patients with low-grade glioma (LGG) corresponds with improved clinical outcome, yet remains a central challenge to the neurosurgical oncologist. Although 5-aminolevulinic acid (5- ALA) induced tumor fluorescence is a strategy that can improve EOR in gliomas, only glioblastomas routinely fluoresce following 5-ALA administration. Intraoperative confocal microscopy adapts conventional confocal technology to a handheld probe that provides real-time fluorescent imaging at up to 1000 magnification. The authors report a combined approach in which intraoperative confocal microscopy is used to visualize 5-ALA tumor fluorescence in LGGs during the course of microsurgical resection. Methods. Following 5-ALA administration, patients with newly diagnosed LGG underwent microsurgical resection. Intraoperative confocal microscopy was conducted at the following points: 1) initial encounter with the tumor; 2) the midpoint of tumor resection; and 3) the presumed brain-tumor interface. Histopathological analysis of these sites correlated tumor infiltration with intraoperative cellular tumor fluorescence. Results. Ten consecutive patients with WHO Grades I and II gliomas underwent microsurgical resection with 5-ALA and intraoperative confocal microscopy. Macroscopic tumor fluorescence was not evident in any patient. However, in each case, intraoperative confocal microscopy identified tumor fluorescence at a cellular level, a finding that corresponded to tumor infiltration on matched histological analyses. Conclusions. Intraoperative confocal microscopy can visualize cellular 5-ALA induced tumor fluorescence within LGGs and at the brain-tumor interface. To assess the clinical value of 5-ALA for high-grade gliomas in conjunction with neuronavigation, and for LGGs in combination with intraoperative confocal microscopy and neuronavigation, a Phase IIIa randomized placebo-controlled trial (BALANCE) is underway at the authors institution. (DOI: / JNS11252) Key Words extent of resection low-grade glioma 5-aminolevulinic acid tumor fluorescence confocal microscopy oncology Maximizing the EOR in patients with LGG improves overall survival, progression-free survival, and malignant progression-free survival. 1,7,18,23,34,36,42,56 Achieving this goal, however, remains a challenge due to the infiltrative nature of these tumors, their similarity in appearance to adjacent normal parenchyma, and their comparatively large size at presentation. 4,18,29,30 In the modern neurosurgical era, technologies such as intraoperative neuronavigation, 19,20,32,52,54,55 intraoperative ultrasonography, 9,26,31,49 and intraoperative MR imaging 2,8,13 15,27,28,39,40 have improved the likelihood of a Abbreviations used in this paper: 5-ALA = 5-aminolevulinic acid; BALANCE = Barrow ALA Intraoperative Confocal Evaluation; BNI = Barrow Neurological Institute; EOR = extent of resection; GTR = gross-total resection; HGG = high-grade glioma; LGG = low-grade glioma; NIHSS = National Institutes of Health Stroke Scale; ROI = region of interest. radiographically complete resection for LGGs. However, although neuronavigation has emerged as a standard of care at most major brain tumor centers, intraoperative ultrasonography and intraoperative MR imaging technologies remain poorly used, due (at least in part) to issues of cost and technical expertise. Nevertheless, despite these technological advances, reported rates of GTR in LGGs during the last decade have remained comparatively low, ranging from 14% to 46%. 11,17,22,23,25,36,41,42,57 Disaggregation of the data sets from these EOR reports for LGG indicates radiographically confirmed GTR for 399 of 1462 LGGs a 27.3% average rate of GTR. A similar analysis of the literature for HGGs, however, reveals a substantially higher rate of GTR (33% 76%), 3,5,6,43,50 and disaggregation of these HGG data sets yields an average GTR rate of 62.3% (1412 of 2266 HGGs). Thus, for the neurosurgical oncologist complete resection of an LGG remains a challenge. 740

2 Tumor fluorescence for low-grade gliomas With the emergence of intraoperative florescenceguided resection performed using 5-ALA, the gap between EOR rates for LGG and HGG will continue to widen. The 5-ALA is an orally administered prodrug that is intracellularly metabolized to form the fluorescent molecule protoporphyrin IX. 10,47 This heme synthesis pathway substrate accumulates preferentially in tumor cells and epithelial tissues and emits a red-violet light (l = nm) when excited with blue light (l = nm). 16,46 Successful neurosurgical integration of 5-ALA induced fluorescence for HGGs was demonstrated by a European randomized controlled trial conducted by Stummer et al. in This Phase IIIa clinical trial was halted following an interim analysis of 270 patients that indicated a 65% versus 36% rate of GTR and a 41% versus 21.1% rate of 6-month progression-free survival benefiting HGG patients who received 5-ALA. Although this study was not powered to assess overall survival, nor were surgeons permitted to use intraoperative neuronavigation during tumor resection, intraoperative 5-ALA fluorescence has since emerged as a valuable adjunct for HGG surgery 21,24,48 in the international neurosurgery community. In the US, its merits remain under investigation, and will be the subject of a prospective, multicenter trial (RTOG 1105). 33,51 For LGGs, however, standard intraoperative 5-ALA fluorescence remains ineffective, because it does not produce visible fluorescence for most tumors. In a handful of cases, heterogeneous tumor fluorescence has been noted in focal areas of anaplastic transformation; however, the vast majority of LGGs are invisible with 5-ALA. 12,16,44,53 Interestingly, protoporphyrin IX fluorescence can be measured ex vivo in LGG tissue following 5-ALA administration. 12,16 In these analyses, the resultant fluorescent intensity of the tumor tissue is significantly higher than in similarly treated normal tissue, 16 and increases proportionally with both tumor grade and MIB-1 proliferative index. 12,47 Taken together, these reports suggest that 5-ALA tumor fluorescence can be microscopically localized to LGGs even when it is not macroscopically evident. Intraoperative confocal microscopy adapts conventional confocal microscope technology to provide highresolution subsurface imaging of living tissue in vivo, allowing visualization of cellular elements and cytoarchitecture. The technology is encased within a handheld, mm, sterilizable, 135 -angled, rigid probe that displays images in real time on an attached external monitor at up to 1000 magnification. A foot switch enables operator control of the focal plane position over a range of 250 μm. Using intravenous fluorescein and topical acriflavine dyes, intraoperative confocal microscopy can detect histological features of glioblastoma in an experimental rodent model, identifying cellular shape and tissue architectural features corresponding to tumor infiltration. 37 In humans, intraoperative confocal microscopy performed using intravenous fluorescein can distinguish the histological features of gliomas, meningiomas, hemangioblastomas, and central neurocytomas in vivo and ex vivo. 35 We report the first combined effort in which intraoperative confocal microscopy is used to visualize 5-ALA tumor fluorescence in LGGs during the course of microsurgical resection. Our initial experience demonstrates the following: 1) integration of intraoperative confocal microscopy and 5-ALA fluorescence into a practical and efficient operating room workflow; 2) detection of 5-ALA fluorescence at a cellular level within WHO Grades I and II LGGs; 3) cellular identification of LGG tumor margins; and 4) equivalence between in vivo and ex vivo use of the confocal microscope when evaluating tumor fluorescence. Methods Patient Selection This study was conducted at the BNI and St. Joseph s Hospital and Medical Center, with approval from the St. Joseph Institutional Review Board (IRB No. 10BN132). An FDA Investigational New Drug approval (No. 109,995) was obtained prior to study initiation. In 2010, 1253 patients with brain tumors underwent microsurgical resection at the BNI, including 352 patients with newly diagnosed or recurrent gliomas. In December 2010 and January 2011, 10 consecutive patients with LGG underwent microsurgical resection combined with 5-ALA fluorescence and confocal microscopy performed by the first author (N.S.). All patients had WHO Grade I or II gliomas, as verified by an attending neuropathologist (J.E. and S.C.) by using standard histological methods and criteria. Study inclusion criteria were as follows: 1) presumed newly diagnosed LGG; 2) patient age 18 years; and 3) normal bone marrow function (white blood cells > 3000, platelets > 100,000). Exclusion criteria were as follows: 1) pregnancy; 2) history of photosensitivity, porphyria, or exfoliative dermatitis; 3) hepatic dysfunction in the last 12 months (defined by aminotransferase [AST], alanine aminotransferase [ALT], alkaline phosphatase [ALP], bilirubin > 2.5-fold normal); 4) plasma creatinine 180 μmol/l; 5) creatinine clearance 60 ml/ min/1.73 m 2 ; and 6) inability to undergo an MR imaging study with intravenous contrast material. Operative Protocol Following informed consent, patients received 20 mg/kg body weight 5-ALA (DUSA Pharmaceuticals, Inc.) mixed with 50 ml of sterile water and orally administered 3 hours prior to surgery. Intraoperatively, all patients underwent routine microsurgical resection with StealthStation Treon (Medtronic, Inc.) neuronavigation. Intraoperative MR imaging was not used in any case. In all cases, Zeiss OPMI Pentero microscopes (Carl Zeiss Surgical GmbH) fitted with Blue 400 fluorescence technology were used. This specialized microscope emits violet-blue excitation light ( nm) through a long-pass filter to enable observation of 5-ALA tumor fluorescence. Using this device, blue fluorescent light was intermittently emitted during tumor resection to assess macroscopic tumor fluorescence. However, the primary guide for resection in all cases was conventional neurosurgical technique with white-light illumination, assisted by neuronavigational guidance. 741

3 N. Sanai et al. Intraoperative confocal microscopy was performed using a handheld confocal endomicroscope probe (Optiscan Pty., Ltd.). In vivo confocal microscopy was conducted during 3 phases of each procedure: 1) on initial encounter of the tumor (based on T2-weighted MR imaging with neuronavigational guidance); 2) at the midpoint of tumor resection; and 3) following resection of the tumor (based on T2-weighted MR imaging with neuronavigational guidance). At each phase, confocal visualization of fluorescent cells prompted image-registered biopsy sampling of the tissue. No biopsy sites demonstrated any evidence of macroscopic fluorescence with Blue 400 fluorescence technology. All excised specimens were then immediately examined using ex vivo confocal microscopy, divided into 2 pieces, and placed either in formalin or in a sterile cryogenic vial and immediately frozen in liquid nitrogen (see Neuropathological Analysis protocol below). Total intraoperative confocal imaging time was approximately 10 minutes per patient. In all cases, microsurgical resection was concluded on the basis of surgical judgment, microscopic visualization, and image guidance. Additional confocal microscopy images were recorded from regions of normal brain, although no biopsy samples were obtained from these areas. Intraoperative Confocal Microscope The unit consists of a miniaturized confocal microscope scanner integrated into a rigid probe connected via a flexible umbilicus to an optical unit and control PC unit. The scanner is based on single-fiber scanning technology in which a single optical fiber acting as both an illumination and detection aperture is used to enact the confocal imaging principle. The fiber is raster-scanned behind a miniature objective lens that projects the scan through a window at the distal end of the probe that is in contact with the tissue. An integrated depth actuator enables the scan to focus to a specific depth in the tissue that is controlled by the operator in 4-μm steps over a range of μm beneath the contact window plane. The scanned field of view is μm, with a lateral resolution of 0.7 μm and an axial resolution (that is, effective optical slice thickness) of approximately 7 μm. One-megapixel scans are collected at frame rates of 0.8 frames/second. The confocal microscope s solid-state laser emits a 488-nm violet-blue excitation light with an incident power of up to 1 mw. Detected light is filtered to the red-violet spectrum via a 605- to 705-nm bandpass filter. Neuropathological Analysis Biopsy specimens were sharply excised under microscopic visualization at each imaging site by using a combination of scalpel and microspatula. Specimens were immediately marked with tissue ink to indicate superior and inferior surfaces, placed on sterile Telfa (Covidien, Inc.), and divided into specimens for formalin fixation and snap freezing. Intraoperative MR neuronavigation was used to record the location of each specimen in 3D coordinates. Subsequently, specimens were cut into 10- μm sections and stained with H & E for histopathological analysis. Care was taken to ensure accurate orientation of the tissue throughout the handling and staining process so that direct comparisons could be made between confocal microscopy images and histological sections from the same area. Central neuropathology review was performed by 2 neuropathologists (J.E. and S.C.) based on WHO guidelines. When appropriate, special stains were chosen to establish a definitive diagnosis. Volume Based on MR Imaging Volumetric measurement of pre- and postoperative imaging was conducted by an independent neuroradiologist who used a previously reported protocol. 36 For all tumors, manual segmentation was performed with ROI analysis to measure tumor volumes (in cubic centimeters) on the basis of FLAIR or T2-weighted axial slices. The EOR was calculated according to the following formula: (preoperative tumor volume - postoperative tumor volume)/preoperative tumor volume. Determination of volumes was made without consideration of clinical outcome. Patient Outcome Measures Preoperatively and at each follow-up appointment, patients underwent neurological examination. Each examination was conducted by 2 clinicians: the attending neurosurgeon (N.S.) and a senior neurosurgical resident. Neurological morbidity was defined by new-onset or worsening deficits related to motor, sensory, visual, language, or cognitive function. Patients with no improvement at their 6-week follow-up visit were considered to have a permanent deficit. When applicable, MR imaging results were also reviewed to confirm that the patients symptoms were not a function of tumor recurrence. In addition to neurological morbidity, the NIHSS was used to quantify and compare neurological deficits preand postoperatively. All patients underwent an NIHSS assessment at their preoperative clinic visit, as well as at 1 week postoperatively. This assessment was conducted by a research nurse (N.H.) certified in NIHSS assessment. Results Patient Demographics and Tumor Characteristics With the aid of a combination of intraoperative confocal microscopy and 5-ALA tumor fluorescence, 10 adult patients with newly diagnosed LGGs were treated by the first author at the BNI in December 2010 and January The patients (6 men and 4 women) had a median age of 35 years (range years). At presentation, most patients had seizures (7 cases), headaches (5), or a focal neurological deficit (5). In 1 case, the lesion was found incidentally following a motor vehicle accident. As shown in Table 1, tumors were most commonly located in the frontal lobe (4 cases), followed by temporal (3), insular (2), and parietal (1) sites. The selection of the operative corridor was dictated by lesion location. Intraoperative cortical and subcortical stimulation mapping was conducted in 4 cases, including 2 awake craniotomies with 742

4 Tumor fluorescence for low-grade gliomas TABLE 1: Demographic data and 5-ALA tumor fluorescence characteristics in 10 patients with LGGs Microscopic Fluorescence Midpoint of Initial Encounter Resection Cavity Margin Age (yrs), Sex Tumor Location WHO Grade Macroscopic Fluorescence Volumetric EOR (%) 19, M frontal I no yes yes yes , F frontal II/III no yes yes yes , M frontal II no yes yes no 98 64, M temporal II no yes yes no 87 39, F parietal II no yes yes yes 99 32, F frontal II no yes yes no 94 38, F temporal II no yes yes yes 92 49, M insular II no yes yes yes 93 24, M temporal II no yes yes no 97 30, M insular II no yes yes yes 90 intraoperative language mapping. The median duration of surgery was 197 minutes (range minutes). Assessment of Cellular Fluorescence All patients received oral 5-ALA preoperatively and underwent intraoperative confocal microscopy at 3 time points during the course of microsurgical resection (see Methods). During each patient s operation, multiple attempts were also made to visualize tumor fluorescence by using the operating microscope s Blue 400 technology. With Blue 400, macroscopic evidence of red-violet tumor fluorescence was not appreciated in any region of any tumor (10 cases). As a control, regions of normal cortex and subcortical white matter that were uninvolved based on radiographic findings were probed with the intraoperative confocal microscope. For each patient, 1 cortical site was selected for in vivo confocal analysis, as were 2 subcortical sites; this equated to 30 presumed normal cortical and subcortical sites among the 10 patients. In these 30 control sites analyzed with the intraoperative confocal microscope, no cellular fluorescence was visualized, even when extending the optical plane 250 μm along the z axis, into the ROI. Additionally, at 5 subcortical sites adjacent to the resection cavity, but without radiographic evidence of tumor on neuronavigation, tissue biopsy samples confirmed the absence of tumor infiltration by using standard histological methods. In conjunction with MR neuronavigation, in vivo confocal microscopy was then conducted on initial encounter of the tumor, at the midpoint of tumor resection, and following resection of the tumor along the cavity walls (Table 1). For all 10 patients, sites assayed on initial encounter with the tumor and at the midpoint of resection demonstrated cellular fluorescence (Fig. 1). Although there was variability in the density of fluorescent cells from region to region within each site of interest, there were no discernible patterns distinguishing one patient s fluorescent confocal imagery from another s. Tumor fluorescence detected with the confocal microscope was not observed to fade during the course of each procedure, nor did its detection require absolute stillness in the field of view. The 10 sites of frank tumor infiltration were then excised and their 3D locations recorded using MR neuronavigation. Immediate ex vivo confocal imaging was then conducted on each specimen while it lay on a strip of Telfa. No rinsing or other preparation of the tissue was required. For these 10 specimens, the ex vivo confocal microscopy results were comparable to corresponding in vivo data in terms of fluorescent cell density, signal intensity, image contrast, and image resolution. Importantly, for both in vivo and ex vivo confocal imaging, tumor fluorescence was only appreciated during the first 100 μm of confocal depth, with no signal detectable beyond this threshold, regardless of the specimen s location within the tumor mass. For each patient, margins of the resection cavities were assessed with intraoperative confocal microscopy once a GTR was thought to have been achieved, based on surgical judgment, microscopic visualization, and neuronavigational image guidance. In 4 of these cases, confocal microscopy confirmed a complete absence of tumor fluorescence along all dimensions of the cavity walls. Within these same regions of confocal image acquisition, standard histopathological studies confirmed an absence of tumor infiltration (8 samples; 2 per patient) (Fig. 2). In the other 6 patients, however, one or more of the cavity walls demonstrated cellular fluorescence (typically > 30 cells per high-power field) on confocal microscopy, despite MR neuronavigation indicating a location beyond the margins of T2 hyperintensity. In each of these 12 fluorescence-positive sites, tumor infiltration was confirmed on the basis of standard histopathological investigation. For 3 patients in whom margins were positive for tumor cells, microsurgical resection of additional layers of tumor revealed underlying tissue planes without evidence of cellular fluorescence on confocal microscopy. In the other 3 patients, however, further microsurgical resection was limited by functional pathways identified with preoperative functional MR imaging (1 patient) or intraoperative subcortical stimulation mapping (2 patients). Postoperatively, all patients demonstrated routine rates of recovery and no evidence of new, permanent neurological deficits. One patient experienced transient dysphasia and 1 patient exhibited symptoms of a supple- 743

5 N. Sanai et al. mental motor area syndrome, although both resolved by 6 weeks postoperatively. The median duration of hospitalization was 3.5 days (range 2 6 days), and there were no reported adverse events related to administration of 5-ALA. The median pre- and postoperative NIHSS scores were 2.1 (range 0 4) and 1.2 (range 0 3), respectively. In 5 patients, symptoms of tumor mass effect causing focal neurological deficit were markedly improved following surgery. No surgical or perioperative complications were encountered. Preoperative and postoperative volumetric MR imaging indicated a median EOR of 95% (range 87% 100%) and a median volume of residual disease of 0.6 cm3 (range cm3). In select cases, a subtotal tumor resection was necessary to preserve functional subcortical pathways identified using preoperative functional MR imaging or intraoperative cortical stimulation mapping. Standard histopathological analysis of the 10 resected tumors indicated the following pathological diagnoses: WHO Grade I dysembryoplastic neuroepithelial tumor (1 lesion), WHO Grade II oligodendroglioma (3), WHO Grade II oligoastrocytoma (1), and WHO Grade II astrocytoma (4). Interestingly, the last patient s tumor was primarily a WHO Grade II oligodendroglioma, but demonstrated pathological foci characteristic of WHO Grade III anaplastic oligodendroglioma. Although 5-ALA fluorescence was seen with confocal microscopy throughout the examined tissue, macroscopic tumor fluorescence was not noted in any region of this patient s tumor, including regions with anaplastic features (Fig. 3). Discussion This initial experience with intraoperative confocal microscopy and 5-ALA tumor fluorescence suggests that a combined approach may expand the utility of 5-ALA beyond glioblastomas. Specifically, we describe 10 consecutive patients with WHO Grades I and II LGGs, including a WHO Grade II oligodendroglioma with histological evidence of anaplastic transformation, that were invisible when we used conventional methods for 5-ALA tumor fluorescence detection. In all patients, however, intraoperative confocal microscopy identified tumor fluorescence at a cellular level, a finding that corresponded to tumor infiltration on matched histological analysis. This combined strategy was equally robust in vivo as it was ex vivo. Taken together, this represents the first successful effort at real-time intraoperative detection of WHO Grades I and II gliomas by using 5-ALA. Although evidence continues to mount in favor of using 5-ALA during HGG resection, the majority of the data suggest that this technique is only reliable when operating on WHO Grade IV gliomas.10,16,21,24,45 48 The most compelling data, however, came from the European 5-ALA Glioma Study Group;45 this study was conducted in the absence of intraoperative neuronavigation, a nearly universal technology at major brain tumor centers. Not surprisingly, the median rate of GTR among the control group for this European Phase IIIa trial was 36%. In contrast, retrospective studies from centers using neuronavigation for HGG resection report a range from 33% to 76%,3,5,6,43,50 equating to an average GTR rate of 62.3%. 744 Fig. 1. Microscopic 5-ALA tumor fluorescence detected in a WHO Grade II glioma by using intraoperative confocal microscopy. A: Axial MR image obtained in a 21-year-old man in whom a right frontal LGG was detected incidentally following a motor vehicle accident. B: Intraoperative view; the handheld confocal microscope was used in the absence of macroscopic 5-ALA tumor fluorescence. C: Intraoperative neuronavigation studies confirming localization of the confocal imaging within the tumor mass. D: Multiple fluorescent cells were observed within this region, corresponding to 5-ALA metabolism. E: Postoperative axial FLAIR MR imaging study confirming a 98% volumetric EOR. Thus, it remains possible that 5-ALA fluorescence will have little impact in improving the EOR for HGG when used in conjunction with neuronavigation and/or other increasingly common guidance techniques, such as intraoperative ultrasonography and intraoperative MR imaging. Nevertheless, this question cannot be answered without a prospective randomized controlled trial combining 5-ALA and intraoperative neuronavigation. Interestingly, for LGGs the potential utility of 5-ALA tumor fluorescence remains more promising, because reported rates of GTR in LGG range from 14% to 46%,11,17,22,23,25,36,41,42,57 equating to an average GTR rate of only 27.3% despite the use of intraoperative neuronavigation. Furthermore, the value of a strategy that enhances LGG visualization is also foreshadowed by the intraoperative MR imaging literature, in which reported rates of GTR are substantially higher (range 50% 91%).2,8,27,28,38,40 Thus, the potential impact of 5-ALA on glioma surgery when combined with neuronavigation and/or intraoperative confocal microscopy remains unclear. To ad

6 Tumor fluorescence for low-grade gliomas Fig. 2. Identification of LGG tumor margins by using confocal microscopy and 5-ALA tumor fluorescence. A: Axial MR image obtained in a 39-year-old woman with a right parietal WHO Grade II glioma who underwent routine microsurgical resection. B: Intraoperative neuronavigation studies identifying a resection cavity margin as radiographically free of tumor. C: Results of intraoperative confocal microscopy at this site were negative, suggesting an absence of infiltrating LGG. D: Histological analysis of this same region, with an H & E stained section confirming the absence of tumor. Original magnification 40. E: Intraoperative neuronavigation studies identifying the presumed margin of the brain-tumor interface. F: Macroscopic 5-ALA tumor fluorescence was not evident, and a region of the cavity wall (box) was analyzed using intraoperative confocal microscopy. G: Image of the same region revealing evidence of persistent fluorescent tumor infiltration. H: Corresponding histopathological analysis of this ROI, with an H & E stained section confirming the presence of infiltrating tumor, including cells with nuclear atypia (arrows). Original magnification 40. I: Postoperative axial FLAIR MR imaging study confirming a 99% volumetric EOR. dress this, we are accruing patients for the BALANCE study. This Phase IIIa randomized placebo-controlled trial consists of 2 study arms with the following goals: 1) assess the efficacy of 5-ALA for HGGs when used in conjunction with intraoperative neuronavigation; and 2) evaluate the impact of 5-ALA on LGG resection when combined with intraoperative confocal microscopy and intraoperative neuronavigation. Importantly, this trial uses the same 5-ALA drug formulation, Gliolan (medac GmbH), as is approved for use in Europe.45 The primary outcome measure for each study arm is volume of residual disease, although neurological morbidity, volumetric EOR, progression-free survival, malignant progressionfree survival, and overall survival will also be assessed. The BALANCE trial will thus determine the clinical value of 5-ALA tumor fluorescence for HGG surgery aided by neuronavigation, and for LGG surgery in which both neuronavigation and intraoperative confocal technology are used. Study accrual is anticipated to conclude in Conclusions Greater EOR for patients with LGG and HGG corresponds with improved clinical outcome, yet remains a central challenge to the neurosurgical oncologist. Although intraoperative adjuncts such as neuronavigation have greatly improved microsurgical efficacy, 5-ALA tumor fluorescence may provide an added advantage. Although newly diagnosed glioblastomas consistently metabolize 5-ALA to fluorescent protoporphyrin IX, tumor fluorescence is less than robust among WHO Grade III gliomas and macroscopically invisible for WHO Grades I and II gliomas. Our initial experience using intraop745

7 N. Sanai et al. clinical impact of 5-ALA for HGGs in conjunction with intraoperative neuronavigation and for LGGs in combination with intraoperative confocal microscopy and intraoperative neuronavigation, a Phase IIIa randomized placebo-controlled trial (BALANCE) is underway at our institution. Disclosure The authors have no financial or marketing relationship with Op tiscan Pty., DUSA Pharmaceuticals, or medac GmbH. Author contributions to the study and manuscript preparation include the following. Conception and design: Sanai, Smith, Spetz ler. Acquisition of data: Sanai, Snyder, Eschbacher, Coons, Honea. Analysis and interpretation of data: Sanai, Snyder. Drafting the article: Sanai, Snyder. Critically revising the article: Sanai, Snyder, Spetzler. References Fig. 3. Intraoperative confocal microscopy visualization of 5-ALA cellular fluorescence in a transforming WHO Grade II/III oligodendroglioma. A: Axial MR image obtained in a 37-year-old woman who presented with new-onset seizures and a nonenhancing right supplementary motor area mass. B: During microsurgical resection, intraoperative neuronavigation confirmed localization of the confocal imaging within the tumor mass. C: Macroscopic 5-ALA tumor fluorescence was not evident, and a region of the tumor (box) was analyzed using intraoperative confocal microscopy. D: Image of the same region revealing 5-ALA induced tumor fluorescence. E: Correlative analysis of this site with an H & E stained section identifying it as a focal region of WHO Grade III histological characteristics, as evidenced by the presence of microvascular proliferation (arrows). Original magnification 40. F: Within this same region, atypical mitotic figures (arrow) were also encountered on an H & E stained section. Original magnification 80. G: Postoperative axial FLAIR MR imaging study confirming a 100% volumetric EOR. erative confocal microscopy to visualize cellular 5-ALA tumor fluorescence suggests that infiltrating tumor cells can be identified within the LGG tumor mass and at the brain-tumor interface. For a definitive evaluation of the Ahmadi R, Dictus C, Hartmann C, Zürn O, Edler L, Hartmann M, et al: Long-term outcome and survival of surgically treated supratentorial low-grade glioma in adult patients. Acta Neurochir (Wien) 151: , 2009 (Erratum in Acta Neurochir (Wien) 161:1367, 2009) 2. Black PM, Alexander E III, Martin C, Moriarty T, Nabavi A, Wong TZ, et al: Craniotomy for tumor treatment in an intraoperative magnetic resonance imaging unit. Neurosurgery 45: , Brown PD, Maurer MJ, Rummans TA, Pollock BE, Ballman KV, Sloan JA, et al: A prospective study of quality of life in adults with newly diagnosed high-grade gliomas: the impact of the extent of resection on quality of life and survival. Neurosurgery 57: , Cavaliere R, Lopes MB, Schiff D: Low-grade gliomas: an update on pathology and therapy. Lancet Neurol 4: , Chaichana KL, Halthore AN, Parker SL, Olivi A, Weingart JD, Brem H, et al: Factors involved in maintaining prolonged functional independence following supratentorial glioblastoma resection. Clinical article. J Neurosurg 114: , Chaichana KL, Kosztowski T, Niranjan A, Olivi A, Weingart JD, Laterra J, et al: Prognostic significance of contrastenhancing anaplastic astrocytomas in adults. Clinical article. J Neurosurg 113: , Chang EF, Smith JS, Chang SM, Lamborn KR, Prados MD, Butowski N, et al: Preoperative prognostic classification system for hemispheric low-grade gliomas in adults. Clinical article. J Neurosurg 109: , Claus EB, Horlacher A, Hsu L, Schwartz RB, Dello-Iacono D, Talos F, et al: Survival rates in patients with low-grade glioma after intraoperative magnetic resonance image guidance. Cancer 103: , Coenen VA, Krings T, Weidemann J, Hans FJ, Reinacher P, Gilsbach JM, et al: Sequential visualization of brain and fiber tract deformation during intracranial surgery with threedimensional ultrasound: an approach to evaluate the effect of brain shift. Neurosurgery 56 (1 Suppl): , Duffner F, Ritz R, Freudenstein D, Weller M, Dietz K, Wessels J: Specific intensity imaging for glioblastoma and neural cell cultures with 5-aminolevulinic acid-derived protoporphyrin IX. J Neurooncol 71: , El-Hateer H, Souhami L, Roberge D, Maestro RD, Leblanc R, Eldebawy E, et al: Low-grade oligodendroglioma: an indolent but incurable disease? Clinical article. J Neurosurg 111: , 2009

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