Intraoperative Confocal Microscopy for BrainTumors: A Feasibility Analysis in Humans
BACKGROUND: The ability to diagnose brain tumors intraoperatively and identify tumormargins during resection could maximize resection and minimize morbidity. Advances inoptical imaging enabled production of a handheld intraoperative confocal microscope.
OBJECTIVE: To present a feasibility analysis of the intraoperative confocal microscopefor brain tumor resection.
METHODS: Thirty-three patients with brain tumor treated at Barrow Neurological In-stitute were examined. All patients received an intravenous bolus of sodium fluoresceinbefore confocal imaging with the Optiscan FIVE 1 system probe. Optical biopsies wereobtained within each tumor and along the tumor-brain interfaces. Correspondingpathologic specimens were then excised and processed. These data was compared bya neuropathologist to identify the concordance for tumor histology, grade, and margins.
RESULTS: Thirty-one of 33 lesions were tumors (93.9%) and 2 cases were identified asradiation necrosis (6.1%). Of the former, 25 (80.6%) were intra-axial and 6 (19.4%) wereextra-axial. Intra-axial tumors were most commonly gliomas and metastases, while allextra-axial tumors were meningiomas. Among high-grade gliomas, vascular neo-proliferation, as well as tumor margins, were identifiable using confocal imaging. Me-ningothelial and fibrous meningiomas were distinct on confocal microcopy—the latterfeatured spindle-shaped cells distinguishable from adjacent parenchyma. Other tumorhistologies correlated well with standard neuropathology tissue preparations.
CONCLUSION: Intraoperative confocal microscopy is a practicable technology forthe resection of human brain tumors. Preliminary analysis demonstrates reliability fora variety of lesions in identifying tumor cells and the tumor-brain interface. Furtherrefinement of this technology depends upon the approval of tumor-specific fluorescentcontrast agents for human use.
Extent of resection is the primary objectivein most brain tumor resections, yet dis-tinguishing normal from abnormal tissue
can only be definitively assessed on postoperativepathologic analysis. For some extra-axial lesions,the gross appearance of the tumor is sufficient toestablish the tumor-brain tissue planes with mi-crodissection techniques. Other lesions, however,are less easily distinguished, particularly in thesetting of prior treatment effect, cerebral edema, ormicroscopic infiltration. This is particularly true for
gliomas and higher-grade meningiomas, wheredefining the extent of resection on the basis of grosstissue characteristics is insufficient and neuro-navigation can be unreliable because of shift. Toovercome these persistent challenges in the re-section of complex intra- and extra-axial braintumors, recent work has been directed towardadapting routine postoperative neuropathologymethods into a real-time intraoperative technique.Beyond identifying tumor margins, the
opportunity to intraoperatively define tumorgrade and histologic subtype is of criticalimportance, particularly for intracranial gliomas,where tumor grade is not reliably predicted witheither preoperative magnetic resonance imaging1
or stereotactic biopsy.2 Intraoperative frozen-section analysis canbe similarly misleading or nondiagnostic, particularly in cases ofmechanical tissue disruption from the resection process.3,4 Suchdiagnostic unpredictability is further complicated by the inherentheterogeneity of gliomas, which can contain high-grade pop-ulations nested within a low-grade stroma.5 Collectively, thesecomplexities represent a significant challenge to the neurosurgicaloncologist, but could be overcome by multiple in vivo opticalbiopsies in the course of tumor exposure and resection.
Confocal microsocopy is an optical imaging technique that usespoint illumination and a spatial pinhole to eliminate out-of-focuslight in specimens that are thicker than the focal plane. In doing so,it enhances optical resolution beyond light microscopy and detectslight produced by fluorescence very close to the focal plane. In-traoperative confocal microscopy is an emerging technology thathas miniaturized this approach to enable visualization of live tissuecytoarchitecture with spatial resolution on a cellular level.6-9 Untilrecently, the size of requisite apparatus limited it to examination ofexcised tissue samples or isolated cells in a bench-top setting. Thelatest incarnation of this technology, however, features fiber-opticand microscopic miniaturization, substantially expanding its por-tability and applicability in an in vivo clinical setting.10-13With the
use of a single optical fiber as both the illumination point sourceand detection pinhole, high-resolution images are acquired andcombined with miniaturized scanning and optical systems.14 Asa consequence, this device has recently been integrated into thedistal tip of conventional video endoscopes and combined withintravenous fluorophores to screen gastrointestinal mucosa forcancer.15,16 The bladder mucosa, skin, and eye have similarly beenstudied with in vivo confocal microscopy.17-20 For brain tumors,we recently used a rodent glioma model to study the capacity ofhandheld confocal imaging to discern microvasculature, the grey-white junction, and tumor margins in the rodent cortex. To date,however, this intraoperative tool has not been applied to humanbrain tissue or pathology. Here, we present a first-look feasibilityanalysis of the intraoperative confocal microscope as an adjunct forhuman brain tumor resection.
METHODS
Ethics Approval
This study was conducted at the Barrow Neurological Institute atSt. Joseph’s Hospital and Medical Center, with experimental approval fromthe St. Joseph’s Hospital and Medical Center’s Institutional Review Board.
FIGURE 1. Protocol for human intraoperative confocal microscopy. A, the locations of all optical biopsies were recorded with 3-dimensional MR neuronavigation. B, multipleregions of tumor were studied with the confocal probe. C, these same regions were then biopsied and examined with standard neuropathology techniques. Used with permission
from Barrow Neurological Institute.
INTRAOPERATIVE CONFOCAL MICROSCOPY FOR HUMAN BRAIN TUMORS
Any patient with an intracranial mass undergoing craniotomy for biopsyand/or resection was considered for entry into the study. Included were allpatients with neurosurgical pathology requiring surgery in which tumorresection might be evaluated by using biopsy and/or resection. This includedpatients with suspicion of brain tumors, such as meningiomas, oligoden-drogliomas, astrocytomas, and metastatic tumors. Patients were excluded ifthey were less than 18 years of age, had an allergy to fluorescein, werepregnant, or were on b-blockers or angiotensin-converting enzymeinhibitors. Preoperative informed consent was obtained for all patients.Intraoperatively, all patients received a 5-mL intravenous bolus of sodiumfluorescein (10% in saline, Pharmalab, Australia) immediately precedinginitiation of intraoperative confocal imaging. Following contrast adminis-tration, the Optiscan FIVE 1 system confocal probe (Optiscan Pty. Ltd.;Notting Hill, Victoria, Australia) was affixed to a Greenberg retractor system(Codman Inc., Raynham, Massachusetts) and the distal tip lowered gentlyonto the surface of the exposed tumor. Care was taken to maintain anorthogonal angle of approach to the target area (Figure 1). Following imageacquisition, the probe was scanned along the presumed tumor-brain in-terface. Several images were acquired at each site and in transition betweensites using a remote foot-pedal control system. All images were stored andcatalogued as digital files. Multiple biopsy specimens were obtained as de-scribed below, and the imaging protocol was then repeated for each spec-imen at increasing depths from the tissue surface up to 500 mm. Additionalconfocal microscope images were recorded from regions of normal brain,although no biopsies were obtained from these areas. In approximately halfof all cases, a neuropathologist was in the operating room at the time ofconfocal microscopy to direct the imaging and diagnostic interpretation.Imaging time was approximately 10 minutes per patient. Total time addedto the duration of each case was 15 to 20 minutes per patient.
Image Acquisition Parameters
The imaging device comprises a miniaturized confocal microscopescanner integrated into a rigid probe connected via a flexible umbilicus toan optical unit and control PC unit which dynamically displayed images inreal time. The probe could be operated as either a handheld unit ormounted onto a Greenberg retractor system with MR neuronavigationintegration for localization of the probe tip. The probe shaft has an outerdiameter of 6.3 mm and a smaller contact window of 5 mm. The scanner isbased on single-fiber scanning technology using a single optical fiber actingas both an illumination and detection aperture to enact the confocalimaging principle. The fiber is raster-scanned behind a miniature objectivelens that projects the scan through a window at the distal end of the probethat is in contact with the tissue. An integrated depth actuator enables thescan to be focused to a specific depth in the tissue that is operator-controlled in 4-mm steps over a range of 0 to 500 mm beneath the contactwindow plane. The scanned field of view was 475 mm 3 475 mm witha lateral resolution of 0.7 mm and an axial resolution (ie, effective opticalslice thickness) of approximately 7 mm. One-megapixel scans were col-lected at frame rates of 0.8 frames/second. In vivo imaging was performedusing the exogenous fluorescent contrast agent sodium fluorescein, asdescribed above. Excitation was by blue laser illumination at a 488-nmwavelength and an incident power from 0 to 1 mW.Detection was filteredto green-yellow light via a 505- to 585-nm bandpass filter.
Tissue Processing and Neuropathological Analysis
Biopsy specimens were sharply excised under microscopic visu-alization at each imaging site using a combination of scalpel and
microspatula. Specimens were immediately marked with tissue ink toindicate superior and inferior surfaces, placed in cassettes, and stored in10% formalin solution. Intraoperative MR image guidance was simul-taneously used to record the location of each specimen in 3-dimensionalcoordinates. Subsequently, specimens were cut into 10-mm sections and
FIGURE 2. Intraoperative confocal images of normal and tumor microvascu-lature. A, normal brain arteriole and capillary systems highlighted with in-
travascular fluorescein. B, multiple branch points are evident and real-time flow
(dark lines) is seen during the course of examination. Used with permission fromBarrow Neurological Institute.
stained with hematoxylin and eosin for histopathological analysis. Carewas taken to ensure accurate orientation of the tissue throughout thehandling and staining process so that direct comparisons could be madebetween confocal microscope images and histologic sections from thesame area. Central neuropathology review was performed by 2 attendingneuropathologists (J.E. and S.C.), based on World Health Organization(WHO) guidelines. All neuropathology specimen preps were charac-terized alongside intraoperative specimen images to enable direct com-parison. When appropriate, special stains were performed to establisha definitive diagnosis.
RESULTS
Patient and Lesion Demographics
This prospective study examined 33 adult patients treated by3 attending neurosurgeons (R.F.S., P.N., and K.S.) at the BarrowNeurological Institute from April 2008 to November 2009. Thisincluded 12 males and 21 females, with a median age of 52 years(range, 19-72). All patients had mass lesions requiring neuro-surgical intervention. The most common lesion location was inthe frontal lobe (n = 18), followed by temporal (n = 6) andparietal (n = 3) sites. Of note, posterior fossa (n = 3) and in-traventricular (n = 2) lesions were also included in the analysis.The selection of operative corridor was dictated by lesion locationand the operative objective, which in most cases was complete
resection. An orbitozygomatic craniotomy was the most commonapproach (n = 17), although retrosigmoid (n = 2), suboccipital(n = 2), and interhemispheric (n = 2) approaches were also used.
Neuropathological Analysis
Overall, 31 of the 33 lesions were diagnosed as tumors (93.9%)and 2 cases were identified as radiation necrosis (6.1%). Of the31 tumors, 25 (80.6%) were intra-axial and 6 (19.4%) wereextra-axial. Intra-axial tumors were most commonly gliomas(n = 21) and metastases (n = 2), while all extra-axial tumors in thestudy were meningiomas (n = 6). Among the gliomas, 8 werehigh-grade (38.1%) and 13 were low-grade (61.9%). Allmeningiomas were WHO Grade I (n = 7) or II (n = 1). Themajority of tumor patients were newly diagnosed (n = 24,77.4%), although a subset had undergone previous surgery andadjuvant therapy (n = 7, 22.6%).An initial examination of tissue integrity during confocal vi-
sualization demonstrated excellent preservation of cellular andsubcellular structures. In both normal and tumor parenchyma,large and small vasculature were evident and intact, both on thetissue surface and as deep as 500 mm to the surface. Within thismicrovasculature, the systolic and diastolic flow of erythrocytetransit was appreciated. In particular, areas of neovascularizationfrom tumor-induced angiogenesis could distinguish tumor fromadjacent normal tissue (Figure 2).
FIGURE 3. Optical biopsy of a human anaplastic oligodendroglioma glioma. A, malignant tumor cells infiltrate the cortical gray matter. Arrow designates neuronal satellitosis.
B, hematoxylin and eosin (5003) staining of a permanent section from this same region, demonstrating prominent neuronal satellitosis. C, intraoperative MR neuronavigation
identifying the exact site of an optical and histologic biopsy. Used with permission from Barrow Neurological Institute.
INTRAOPERATIVE CONFOCAL MICROSCOPY FOR HUMAN BRAIN TUMORS
Intracranial gliomas were the most common pathology en-countered in the study. Among WHO grade III and IV gliomas(n = 7), vascular neoproliferation seen on confocal imaging en-abled both the surgeon and neuropathologist to intraoperativelyidentify abnormal tissue. In all cases, these areas of suspicioncorrelated well with both neuronavigational imaging and histo-logic evidence of tumor. High-grade human glioma specimenswere associated with specific confocal features, including neo-vascularization, dense cellularity, and irregular cellular phenotypes
(Figure 3). Waves of varied density, likely corresponding to re-gions of necrosis, were also noted. Importantly, these featureswere evident to both the surgeon and neuropathologist, allowingfor quick integration of confocal microscope findings into theoperative plan. Low-grade gliomas were similarly distinctive withintraoperative confocal microscopy. Although neovascular pro-liferation was less evident, distinctions in cell density and cellularmorphology corresponded with T2-weighted signal abnormal-ities on MR imaging. Specimens analyzed from regions where
FIGURE 4. Optical biopsy of a human central neurocytoma. A, confocal imaging at the tumor margin, demonstrating a gradual transition from a cell-denseregion of tumor (left) to normal parenchyma (right). B, intraoperative MR neuronavigation identifying the exact site of an optical and histologic biopsy.
C, hematoxylin and eosin staining of a permanent section, demonstrating clear tumor infiltration. D, intraoperative photograph of the retractor-mounted
confocal microscope probe examining tumor tissue through an interhemispheric corridor. Used with permission from Barrow Neurological Institute.
a confocal optical biopsy was taken all demonstrated diagnosticpatterns of low-grade histology on permanent sections. Impor-tantly, of the 5 pure oligodendrogliomas in this study, eachdemonstrated a pattern on confocal microscopy distinct fromother astrocytomas of comparable grade. However, mixed oli-goastrocytomas (n = 3) did not appear to have a unique signatureon intraoperative imaging and could not be reliably distinguishedfrom low-grade astrocytomas.
Other intra-axial lesions also demonstrated unique confocalsignatures associated with their histologic phenotype. For centralneurocytomas (n = 1), in vivo imaging demonstrated uniformround cells (Figure 4) organized in a honeycomb conformation,embedded against a background of arborized capillaries. He-mangioblastomas (n = 1) were similarly distinctive on confocalimaging and were identified by large stromal cells mixed witha dense capillary network. Within these tumors, both cysticchanges and areas of microhemorrhage could also be appreciated.
Meningiomas were the primary extra-axial lesions characterizedin this study. Fluorescein-enhanced confocal images of these tu-mors highlighted fine histopathological detail. For the classic me-ningothelial meningioma (n = 5), tumor cells were largely uniformin configuration and organized as dense sheets of cells withoutevidence of whorls or psammoma bodies. In contrast, fibrousmeningiomas (n = 1) contained cells with spindle-shaped mor-phology, easily distinguished from adjacent normal parenchyma
(Figure 5). These tumors also contained fibrous bundles of matrixinterlacing with adjacent tumor cells and dura. Although onemeningioma demonstrated WHO grade II histology, its cellularfeatures were indistinct from others on confocal microscopy.However, the tumor-brain interface of this lesion did appear morepoorly defined compared with borders surveyed in other WHOgrade I meningiomas.
DISCUSSION
Our initial feasibility analysis of the intraoperative confocalmicroscope establishes its potential value during the microsurgicalresection of both intra- and extra-axial tumors. For a variety oftumor histologies, including gliomas, meningiomas, hemangio-blastomas, and central neurocytomas, this handheld devicegenerates a real-time, fluorescein-enhanced pathologic image thatis of sufficient resolution for a neuropathologist to establisha preliminary diagnosis (Figure 6). With mounting evidence inthe literature supporting the value of extent of resection for manyintracranial tumors, including gliomas,21 several intraoperativetechniques have been developed to this end. These include in-traoperative ultrasound,22 intraoperative MRI,23 optical spec-troscopy,24 and fluorescent dyes such as 5-aminolevulinic acid.25
However, none of these technologies can detect infiltrating tumormargins at a microscopic level. In contrast, our preliminary
FIGURE 5. Optical biopsy of a human low-grade meningioma. A, proliferation of neoplastic cells with predominantly elongated nuclei and prominent collagen deposition
(arrow). B, standard hematoxylin and eosin staining (2003) of a permanent section from this same region, confirming WHO grade I histology. C, intraoperative MRneuronavigation identifying the exact site of an optical and histologic biopsy. Used with permission from Barrow Neurological Institute.
INTRAOPERATIVE CONFOCAL MICROSCOPY FOR HUMAN BRAIN TUMORS
analysis of intraoperative confocal microscopy not only suggestsa correlation between imaging and tumor grade, but demon-strates a capacity to distinguish tumor margins from adjacentparenchyma.
Tumor heterogeneity and biopsy sampling error remain a con-siderable source of inaccuracy and a primary cause of gliomaundergrading. The ease with which a handheld confocal micro-scope generates numerous optical biopsies during the course oftumor exposure and resection may effectively neutralize such di-agnostic imprecision. Furthermore, the ability of the device to scanas much as 500 mm through the visible tissue surface permitsanalysis of an even broader spectrum of tissue. This unique ca-pability also raises the possibility of examining subependymal re-gions through an intraventricular corridor, allowing intraoperativeconfocal microscopy to detect pockets of subependymal tumormigration that are radiographically undetectable and remote fromthe primary tumor site. It has been postulated that subependymalspread is both a negative prognostic sign and a primary route ofglioma migration.26 The ability to detect its occurrence at an earlystage may permit anticipatory and focused interventions, as well aslead to insight into basic mechanisms of gliomagenesis.27
Routine neuropathological diagnostics depend on excision andprocessing of tumor tissue, which can be both mechanically andchemically altered during this process. Use of intraoperativeconfocal microscopy circumvents these limitations with real-
time in situ visualization of tumor cytoarchitecture and micro-vasculature. The opportunity to observe active blood flowthrough tumor capillaries also provides a unique visual dimen-sion to tissue assessment. It remains unclear what impact anti-angiogenic agents may have on the ability to identify tumorneovascularization, although angioarchitecture is one of severaldistinguishing features enhanced with confocal microscopy. Theprobe can also quickly survey the visible extent of a tumor withthe potential to detect disconnected, cell-dense islands that mayportend worse histologic grade and heterogeneity. Additionalpilot studies will not only enhance our familiarization with thistechnique, but may demonstrate an extent of correlation withfrozen-section findings that could allow its eventual replacementof conventional intraoperative diagnostic methods. This mod-ernized approach to intraoperative diagnosis would also lenditself to remote, Internet-based analyses, expanding the accessi-bility of diagnostic expertise beyond centers with dedicatedtumor neuropathologists.Beyond its utility for tumor diagnosis, intraoperative confocal
microscopy also provides a real-time alternative to neuro-navigation in identifying abnormal tissue. This aspect is partic-ularly useful in cases of brain shift, where the parenchymallandmarks no longer colocalize with MR images obtained pre-operatively. In these circumstances, confocal microscopy canconfirm the presence of tumor when the reliability of
FIGURE 6. Optical biopsy of a human anaplastic astrocytoma. A, region of increased cellularity, pleomorphism, and nuclear atypia. B, standard hematoxylin and eosin
staining (5003) of a permanent section from this same region demonstrating comparable observations and suggesting a WHO grade III glioma. C, intraoperative MR
neuronavigation identifying the exact site of an optical and histologic biopsy. Used with permission from Barrow Neurological Institute.
neuronavigation is in doubt. Future studies comparing the pre-dictive value of confocal microscopy with intraoperative MRimaging and intraoperative ultrasound techniques may be of valueas well.
Despite these promising findings, our initial effort to applyconfocal microscopy to human brain tumor resections revealedseveral areas needing technical refinement. Because of its relianceon optics, the current technology is susceptible to erythrocytecontamination of the probe, which can obscure the field of view.In this study, contamination was addressed by elevating the probefrom the tissue surface and manually cleaning it with gentleirrigation. Future versions of the device may benefit from anautoirrigation mechanism, similar to the endoscope. Further-more, while fluorescein contrast allows for detailed assessment ofoverall tissue cellularity, the ability to discern cellular cytoplasmremains limited, as does its specificity for nuclear morphology.Since tumor cell nucleus-to-cytoplasm ratios are critical to his-topathological diagnosis, other contrasting agents should be in-vestigated. One candidate agent is 5-aminolevulinic acid, whichallows highly specific localization of tumor cells, but currently isonly available for clinical use in Europe.
CONCLUSION
This initial analysis of intraoperative confocal microscopy inhumans demonstrates its feasibility in examining a variety of intra-and extra-axial brain tumors. A preliminary assessment of thetechnology demonstrates its capacity not only to identify tumor cellpopulations in situ, but also potentially to replace the practice offrozen intraoperative specimen collection. As such, the techniquemay compliment conventional neuropathological diagnostictechniques, while reducing operative time and increasing samplesize, particularly at the tumor margins. Intraoperative confocalmicroscopy also offers the unique ability to directly examine hu-man brain tumor biology in vivo, without relying on xenograftanimal models or surrogate imaging. Taken together, this pre-liminary study highlights a potentially useful new intraoperativeparadigm, but one that requires further study and refinement.
Disclosure
The Barrow Neurological Institute and authors of this publication have no
financial or marketing relationship with Optiscan Pty. Ltd. The authors have no
personal financial or institutional interest in any of the drugs, materials, or devices
described in this article.
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Intraoperative optical spectroscopy identifies infiltrating glioma margins with high
In this exciting technological advance, the authors use intraoperativeconfocal microscopy to provide intraoperative in situ microscopic
evidence of tumor. The article thus shows that this is feasible in humans.This prospective study was not designed to answer the question ofsensitivity and specificity of this technology with regard to finding areasof tumor. Instead, it was designed to inform the readers in the neuro-surgical community that the instrument could be used. With a total of33 patients with brain tumors enrolled in the study, the authors provide
excellent evidence and experience, showing that it is possible to use thistechnological adjunct. One concern may be that this study could bepossibly utilized by a company to market intraoperative confocalmicroscopy before a prospective trial to determine the tool’s sensitivityand specificity is conducted. Notwithstanding this concern, this is an-other example of creative neurosurgeons using their scientific ingenuityto discover new ways to remove even microscopic remnants of tumors.
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