Next, the dura was cut open and reflected away from the cortical surface. The NIR camera was then positioned above the cortex. NIR imaging identified fluorescence in 12 of the 15 (78%) patients. The mean SBR in these cases increased to 7.5 ± 0.93 (77% of final SBR), ultimately improving to a mean 9.5 ± 0.8 (range, 5.5-14.8, representing 100% of final SBR) upon final exposure of the tumor (Figure 2, Table 3). Thus, the fluorescent signal was visible before opening the dura (61%), became stronger at the cortical surface after dural opening (77%), and demonstrated a strong (100%) SBR upon finally exposing the tumor via corticectomy.
Total NIR imaging time did not exceed 12 minutes in any patient.
NIR Fluorescence Is Sensitive But Not Specific for Residual Tumor at Surgical Margins
In addition to gross examination of the tumor mass, we studied the surgical margins during and at the completion of the surgical resection. After the senior surgeon deemed the surgery to be complete, the fluorescent camera was used to detect potential areas of residual disease. The NIR imaging device was used to scan the wound bed, and areas of residual fluorescence were biopsied as deemed safe based on adjacent anatomy. Diagnostic test characteristics were calculated concentrating only on the 12 patients with contrast-enhancing gliomas. Seventy-one specimens (12 bulk tumor specimens plus an additional 59 margin biopsies) were analyzed.
Of the 71 specimens in all enhancing tumors, 51 (71.8%) demonstrated glioma (tumor) tissue based on final histopathology and 61 (85.9%) were positive for NIR signal (see 2 × 2 contingency tables in Table 4). Using tumor on final pathology as the gold standard, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) based on the surgeon's impression (visible light) alone, values were (respectively) 84.3%, 80%, 91.5%, 66.7%. In contrast, the sensitivity, specificity, PPV, and NPV of NIR intraoperative imaging for identifying tumor was (respectively) 98%, 45%, 82%, and 90% (Table 4A and 4B). Of the 11 samples with false-positive NIR signal, 2 specimens demonstrated no specific pathological change in normal brain, 3 specimens demonstrated necrosis, 4 specimens demonstrated reactive gliosis and atypical cells, 1 specimen demonstrated normal choroid plexus, and 1 specimen consisted of scar and fibrous tissue from prior surgery.
As a subset analysis, we calculated the test characteristics of NIR imaging for newly diagnosed GBM separately. Eight patients were diagnosed with new GBM, and a total of 34 specimen biopsies were performed. Twenty-seven (79%) were positive for tumor based on pathological examination and 30 (88%) specimens were positive for NIR signal. Using the presence of tumor on final pathology as a gold standard, the sensitivity, specificity, PPV, and NPV based on the surgeon's impression and visible light alone were (respectively) 85.2%, 100%, 100%, and 63.6%. In contrast, the sensitivity, specificity, PPV, and NPV of NIR intraoperative imaging for identifying tumor were (respectively) 96.3%, 42.9%, 86.7%, and 75% (Table 4C and 4D). Of the 4 specimens with false-positive NIR signal, the pathology consisted of 1 specimen containing choroid plexus, 1 specimen demonstrating reactive gliosis, and 2 specimens demonstrating normal brain without specific pathological change. Of note, in contrast with experience in 5-ALA, we did find normal choroid plexus to retain ICG but did not see similar retention of dye in normal ependymal lining.
In order to determine the utility of NIR imaging to achieve a gross total resection, we performed a retrospective analysis of the data. Of note, we intentionally specified in our study protocol that the extent of surgery would not change based on the NIR results, and as such we do not believe we can adequately address the question of extent of resection with this current study. However, we did look at the postoperative MRI for a gross total resection as seen in Table 2. We correlated this with the presence of true negative margin biopsy specimens. Of the 12 enhancing gliomas, 4 patients had true-negative biopsy specimens and all 4 had gross total resection seen on MRI. In contrast, of the 12 enhancing gliomas, 8 patients did not have true-negative margin biopsy specimens and only 3 of these had gross total resection on postoperative MRI. This is suggestive of the benefit of a true-negative NIR signal after resection.
Better visualization (surgical microscope), intraoperative MRI and imaging-based navigational systems (with or without technologies to account for brain shift) have improved the ability of neurosurgeons to identify tumor and to distinguish it from normal tissues. Fluorescent contrast agents take visualization to another level with the potential for real-time imaging and tumor/cell-specific identification of disease. 8,9,12,16,24 Although 5-ALA is not FDA approved for use in tumor imaging, surgical resection of glioma using 5-ALA has been favorably demonstrated through multiple studies, as best summarized by Zhao et al. 2 The goal of this preliminary study was to determine if Second Window ICG delivery and NIR imaging could identify brain tumors during a craniotomy. In this pilot study, 15 patients with intracranial glioma underwent systemic injection of 5 mg/kg ICG. NIR signal detected 80% of gliomas, suggesting colocalization of the ICG to the tumor. The presence of tumor NIR fluorescence was independent of tumor size, histology, and time from dye injection to tumor imaging. This study had several important findings.
First, ICG has been in the armamentarium of the vascular neurosurgeon for well over a decade. 10,25 ICG is rapidly cleared from the circulation (t1/2<10 minutes), and thus has been used to develop contrast based on perfusion to tumor vs normal brain parenchyma. For example, ICG has been used to identify peritumoral vasculature26,27 with low parenchymal uptake demonstrated. 28 The resection of hemangioblastomas, a vascular tumor, mediated by ICG has been described in the brain27 and in the spinal cord29 as case reports. These studies aimed to visualize ICG within minutes of administration, using a low relative dose, and as such focused on ICG as a videoangiography tool. However, the use of ICG in the delayed phase as a “second window ICG” technique as a tumor-specific contrast agent is not intuitive.
Other investigators have previously studied ICG to visualize brain tumor parenchyma in a rodent model. 30,31 Haglund et al, 32 for example, demonstrated in a rat glioma model that ICG remained within tumors with relatively few glioma cells seen beyond the ICG-laden tumor margin. The doses utilized ranged from 60 to 120 mg/kg with imaging taking place at least 1 hour following ICG administration. 30,32 The dose utilized was above the 50% lethal dose (LD50) of ICG, and, thus, it was not practical for human use.
In this study, we administered Second Window ICG at 5 mg/kg, which is below the LD50 of ICG, and is safe for human use. We did not experience any significant acute toxicity at this elevated dose. Although ICG for videoangiography and ICG for “Second Window ICG” tumor visualization use the same chemical formulation (C43H47N2O6S2.Na), we chose to name this technique “Second Window ICG” to draw attention to 2 key factors: (1) the higher dose was solubilized in sodium chloride, and (2) the drug was not used as a videoangiography agent but as a small-molecule contrast agent based on the EPR effect. Despite rapid clearance of ICG from the vasculature (half-life less than 10 minutes), sufficient ICG remained within gadolinium-enhancing intracranial gliomas 24 hours after injection; this results in measurable NIR fluorescence in the operating room.
The optimization of the Second Window ICG technique has been explored in a prior publication by Jiang et al. 18 In this article, a syngeneic murine flank tumor model was used to test NIR imaging of ICG at various doses ranging from 0 to 10 mg/kg. Importantly, NIR imaging was performed serially from minutes to 72 hours after administration. The vascular visualization was contrasted with tumor visualization, and over time the videoangiography visualization dissipated, whereas the tumor visualization improved, peaked at 24 hours, and then dissipated as well. The optimal imaging of tumor rather than vasculature for “Second Window ICG” was seen at doses ranging from 5 to 10 mg/kg and tumor signal was best appreciated at 24 hours. Having developed this dose and timing schema in rodents, 10,16,17 we now extend this work to intracranial tumors in this publication. The gross observation that 12 of the 15 patients' brain tumor specimen retained measurable NIR signal parallels the experience previously published in lung cancer, 8,10,16,17 and leads us to the tentative hypothesis that gadolinium-enhancing gliomas with inherently leaky vasculature can be visualized with a NIR camera by taking advantage of the EPR-mediated delivery of Second Window ICG. Given that ICG is delivered via passive diffusion, we posit that it remains extracellular in keeping with prior studies. 25,31,33 We have tentatively termed this technique “DEPIcT” which stands for “Delayed Enhanced Permeability and retention of near Infrared Contrast dye for Tumors.” The results from this cohort of patients demonstrate that Second Window ICG can be delivered systemically to provide NIR fluorescence in brain tumors at high signal strength (approximately 9 times higher than background). When delivered at the timing and dose specified, contrast-enhancing brain tumors demonstrated retention of Second Window ICG based on real-time fluorescence.
The second major finding in this study was tumor localization by real-time, NIR imaging provides similar information as preoperative MRI scanning data. Current neuronavigation techniques allow neurosurgeons to make judgments between preoperatively acquired MRI scans and areas felt to represent tumor in the operating room.34 However, even if the coregistration of the surface anatomy of the skin is precise, the brain can shift. Osmolar therapy/diuresis, opening of cerebral cisterns, and corticectomy/resection/tumor cavity deformation all create major challenges for intraoperative navigational accuracy. In addition to brain shift, increased setup times, and increased operative duration, its indirect methods of visualization detract from the known benefits of these technologies. 35 Because of this situation, many centers have used an intraoperative MRI scanner within the operating room environment, usually of lower magnetic field strength in order to improve safety and maneuverability. Although intraoperative MRI can take account for brain shift (during the singular time of image acquisition) the timing, dose, and type of intraoperative contrast agent used may affect which areas of the tumor (or brain) are ultimately resected. Intraoperative MRI remains an expensive technology in terms of cost and time. 35 These shortcomings continue to fuel interest in systemically delivered fluorescent compounds that produce a high SBR within tumors in real time. 36
The third major discovery in this series was the value of NIR imaging (λexcitation > 780 nm). At present, the FDA in the United States and the European Medicines Association have approved only a handful of systemic fluorescent contrast agents for use in humans. ICG remains the only NIR fluorescent contrast agent that has been approved for systemic injection in humans. 37 The primary value of NIR imaging over other optical techniques is the ability to obtain superior depth of penetration and less autofluorescence and background noise from the surrounding cranium and brain parenchyma. 10 Because of its longer wavelength, we were able to visualize Second Window ICG accumulation in enhancing gliomas even through the dura and through presumably uninvolved cortical parenchyma up to a depth of 13 mm. This technique allows us to localize intracranial glioma without opening the dura in real time (Figures 4-6).
A fourth major finding from this study was our ability to detect margins. We hypothesized that NIR intraoperative imaging in real time may be more sensitive than white light alone in identifying brain tumors. Indeed, we found that, in the identification of gliomas, there appeared to be a trend toward improved sensitivity of NIR at the expense of specificity compared with white light alone. Similarly, the NPV was improved at the expense of PPV with the use of NIR fluorescence. In our data set of 71 samples of contrast-enhancing gliomas, the surgeon's sensitivity/specificity/PPV/NPV was 84.3%/80%/91.5%/66.7% compared with visualization mediated by NIR fluorescence (sensitivity/specificity/PPV/NPV of 98%/45%/82%/90%). The test accuracy as determined by the area under the curve appeared to be better for visible light alone vs NIR, 0.82 vs 0.72. This is most likely because of the low specificity of NIR signal. By comparison, 5-ALA has a summary ROC curve at 94% based on meta-analysis; pooled analysis in patients with GBM who underwent surgery with 5-ALA yielded a sensitivity of 87% and specificity of 89%.2 At this point, the use of Second Window ICG in the detection of tumor margins for oncologic control remains an area requiring further work. Hence, its use for detection of tumor margins for oncologic control remains an area requiring further work.
Although the test characteristics of Second Window ICG administered 24 hours to visualization demonstrate low specificity, 5-ALA, the only fluorescent drug in widespread use for glioma surgery, also demonstrates limitations as a diagnostic test. Valdes et al38 studied the test characteristics of 5-ALA fluorescence imaging and calculated a sensitivity of 47% and specificity 100% in a heterogeneous group of 14 patients. In a follow-up study focusing on 11 patients with newly diagnosed GBM (124 biopsy specimens), Roberts et al39 calculated the test characteristics of 5-ALA. Of the 124 specimens biopsied, 70% were positive for tumor, and thus 30% were not positive for tumor. The sensitivity of 5-ALA without the spectrophotometer but using the surgical microscope was 75%, with a specificity of 71%, PPV of 95%, and a NPV of 26%.40 Similarly, work by Stummer et al4 calculated a PPV of 82.8% and a NPV of 40% in a group of 29 patients with GBM (318 biopsy specimens). A recent meta-analysis published by Zhao et al2 has pooled data together and has concluded that 5-ALA has an overall sensitivity 87%, specificity 89%. Given that 5-ALA does not appear to be >90% sensitive or specific, we publish this article in an effort to explore ways that Second Window ICG, a nonspecific NIR dye, may improve glioma surgery.
The last major finding that warrants discussion is the correlation of NIR imaging and the preoperative MRI scanning. ICG is not receptor specific. Within the circulation, ICG binds to plasma albumin; we suspect that at high doses it remains trapped within the extracellular matrix of tumor tissue. As such, it is most likely not receptor bound or receptor specific. 23,41 We are not surprised by the relatively low specificity with this technique, because ICG is not receptor specific. 2 We posit that Second Window ICG as an intraoperative tumor imaging adjunct may be useful for initial localization of gliomas because it correlates with contrast enhancement on MRI, thus allowing the surgeon to “see” the contrast-enhancing portion of the tumor early, oftentimes before dural opening. Indeed, as seen in Figure 3, the degree of contrast enhancement appears to be directly correlated with the NIR signal, implying that the early amount of accumulation of gadolinium within a tumor correlates with the late (approximately 24 hours later) accumulation of Second Window ICG within the tumor. Indeed, gadolinium enhancement has been used as a proxy for disease in high-grade gliomas, 40 with a diagnostic sensitivity (T1 contrast enhancement alone) of 72.5%, specificity of 65%, PPV of 86.1%, and NPV of 44.1%. The ROC characteristics are increased further with the interpretation of multiple sequences including diffusion and cerebral blood volume. 42 Although we now understand that tumor cells in gliomas may exist beyond the enhancing margins and into surrounding fluid-attenuated inversion recovery/T2 hyperintense regions, the primary aim continues to be to achieve as complete a resection of the enhancing portions of these tumors, as possible, while maintaining function. 43-46
This pilot study demonstrated 1 major limitation to NIR imaging for brain tumors. NIR visualization of Second Window ICG does not appear to be specific for glioma. It is possible that areas of adjacent edema or inflammatory change passively accumulate ICG by diffusion or by increased vascularity. Instead, we hypothesize that NIR visualization of Second Window ICG in delayed (24-h fashion) appears to best correlate with gadolinium enhancement. For high-grade gliomas, this may provide value for surgical resection, because the general goal is maximal resection of the contrast-enhancing portion of the tumor. In contrast, for grade II gliomas that generally do not enhance with gadolinium, Second Window ICG as administered via DEPICT technique may not provide any significant value.
We acknowledge that there are many caveats to this study. First, this is a pilot study; therefore, the study size is small (n = 15). In addition, SBR is an arbitrary number and may not be a true measure of tissue fluorescence. In addition, the SBR may vary with time after administration. Thus, correlating metabolism to fluorescence may not accurately represent the biology of the nodule. The test characteristics obtained in this study are largely determined by the number of biopsies taken at the time of surgery, and thus accurate test characteristic calculation is limited. Because of the limited number of biopsy samples, we combined the margin specimens with the bulk tumor specimens to maximize the power of this analysis, but of course this has the drawback of limiting the ability to draw conclusions regarding the utility of margin detection.
Another important caveat of this Second Window ICG technique of visualization is the difficulty in interpretation of the image on the screen. The VisionSense Iridium system (Visionsense) provides a 2-dimensional view of a 3-dimensional scene. This handicap is also coupled to the concern that NIR is a longer wavelength, and, thus, the NIR signal may actually be slightly deeper or even behind normal brain parenchyma or even dura. Hence, the screen may show a bright object that is actually a few millimeters behind normal parenchyma. Careful dissection and understanding of the physical properties of the NIR signal and the 2-dimensional screen must be made by the trained observer before embarking on further surgical resection. We plan to investigate issues of parallax in 3 dimensions in future work.
In conclusion, ICG is one of several NIR fluorescent agents, but it is the only NIR fluorescent contrast agent approved for use in humans by the FDA. 37 It is low in cost, minimally toxic, and provides for real-time visualization. This is the first work to demonstrate practical sensitivity using Second Window ICG as an optical contrast agent during the neurosurgical resection of human brain tumors. The method of drug delivery used in this technique relies on passive diffusion. We demonstrate that gadolinium-enhancing tumors show measurable NIR contrast in the operating room with real-time visualization. The degree of gadolinium enhancement on the preoperative MRI appears to correlate with the NIR signal intensity within the tumor. The sensitivity of this technique appears to be better than bright light alone but at the expense of specificity. Second Window ICG may provide a practical and sensitive means of identifying glioma before dural opening, and before corticectomy for accurate localization. Its use for margin detection and establishing the extent of resection remains under study.
Dr Lee owns stock options in VisionSense. Dr Singhal holds patent rights over technologies presented in this article. Supported in part by the National Institutes of Health R01 CA193556 (SS), and the Institute for Translational Medicine and Therapeutics of the Perelman School of Medicine at the University of Pennsylvania (JYKL). In addition, research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR000003 (JKYL). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The other authors have no personal, financial, or institutional interest in any of the drugs, materials, or devices described in this article.
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The authors present a timely pilot study on “Second Window ICG,” repurposing of ICG for intraoperative real-time detection of gliomas. Although many questions about this agent's efficacy still remain, the authors have demonstrated sensitive high uptake and retention of the fluorophore correlated with gadolinium-contrast MRI of tumor in a small cohort of 15 glioma patients. This approach is innovative repurposing of an already approved agent for human use, and there is a low barrier of entry because of an excellent safety record, available detection apparatus, long-imaging window, and experience in standard clinical use for vascular procedures. However, further work is necessary to study “Second Window ICG,” to evaluate its efficacy and limitations compared with other fluorescent dyes such as fluorescein and 5-ALA. If the validation studies are completed successfully, these nonspecific dyes may be useful adjuncts in optical surgical oncology applications. Moreover, this would be a significant step toward future successful development, translation, and regulatory approval of even more advanced cancer-targeted and tumor-specific agents and technologies.
Ray R. Zhang
Jamey P. Weichert
John S. Kuo
The authors provide a proof of concept that ICG can be used to visualize gliomas intraoperatively. Twenty-four hours (“second window”) after ICG administration the fluorophore, accumulated in contrast-enhancing gliomas, is detected with a separate special camera system, brought in above the surgical field. Of special note is that using wavelength in the near-infrared spectrum, depth penetration of up to 13 mm below the surface is achieved. Further refinement and appropriate clinical evaluation for safety and efficacy are needed to determine whether this innovative technique is merely colorful or also helpful.
However, the described method illustrates that the spectrum of light has more potential (ie, multispectral analysis and penetration to various depths) but is presently not fully utilized to visualize the brain and its pathologies. Light provides more information than our eyes can detect. In order to use the full spectrum, without constantly changing cameras, digital image processing has to be improved and integrated directly into our surgical microscopes.
Investigating this avenue may bridge the gap between surface-visualization, as in fluorescence-guided resection (eg, 5-ALA), and depth visualization (intraoperative MRI, CT, US) as the current standards for intraoperative imaging. Articles like the current one open this avenue.
The authors are to be commended and encouraged to continue their investigations. As intriguing and beautiful as optical imaging techniques are, the real test remains: to prove that they provide practical information for surgical decision making toward our ultimate goal, improving our patients' treatment.
Even in the context of infiltrative, high-grade gliomas, maximal safe resection carries a clear survival benefit for patients, and, in this regard, intraoperative surgical adjuncts to achieving “gross total resection” as measured by postoperative MRI have potential clinical utility. The authors describe an interesting and novel variation on the fluorescent dye ICG, named Second Window ICG, which enables visualization of contrast-enhancing tumor through a near-infrared range (NIR) camera. This preliminary study is carefully performed, with pathology correlates for their NIR fluorescence technique in a number of samples, and overall demonstrates that, although their dye is not tumor specific, the Second Window ICG signal correlates well with contrast enhancement on MRI. The delays in US access to 5-ALA and the cost-prohibitive nature of intraoperative MRI capability for many institutions create a real opportunity for intraoperative tumor visualization techniques such as this in the brain tumor space. Integration of their technique with an operating microscope or endo/exoscope and additional data regarding the interpretation of what “positive” Second Window ICG signal represents—especially since NIR signal can be seen through “normal” tissue—are interesting points for future investigation.
Albert H. Kim
St. Louis, Missouri
Keywords:Copyright © by the Congress of Neurological Surgeons
Brain tumor; Fluorescence; Indocyanine green; Near-infrared