I'd like to welcome you all to our webinar entitled Current Applications and Advances in Fluorescence-Guided Surgery. My name is Costas Hadjipanais. I will be directing this webinar today. It is sponsored by the American Association of Neurological Surgeons in addition to NREF. Here are my disclosures. I would like to remind the attendees to our webinar that 5-ALA is an investigational drug and not FDA-approved. It is used under an IND in this country. Fluorescein sodium will be discussed as well and it's used off-label. This complimentary webinar is presented by the Neurosurgery Research and Education Foundation with support from Leica Microsystems. I'd like to remind the viewers that it does contain content presented by the NREF. Leica's sponsorship of this webinar does not imply Leica's recommendation or endorsement of the content of the webinar. Leica does not make any representational warranty, but the content of the webinar weighs all responsibility for any content provided by the webinar. The agenda for the webinar will be as follows. We will discuss why fluorescence-guided surgery for brain tumors. We will then move on to current fluorescence-guided surgery technologies, including fluorescence-guided surgery in tumor types in the brain and spinal cord. Discussion of the diagnostic accuracy of fluorescence-guided surgery will be reviewed in addition to level one evidence supporting fluorescence-guided surgery. Current extent of resection studies will be discussed in addition to patient safety with fluorescence-guided surgery. Neurofluorescence-guided surgery technologies will then be followed, and the webinar will conclude with the current regulatory state of fluorescence-guided surgery in the United States. So why fluorescence-guided surgery for brain tumors? Well, we know that improved intraoperative visualization of brain tumors helps with surgery. The ability to visualize the tumor in real time provides direct image guidance of the tumor, and it also allows us to visualize the tumor independent of brain shift and neuronavigation, which we rely on conventional methods. Direct visualization of the tumor also permits more extensive resection of the malignant brain tumor with infiltrative biology, and we believe that safer resection of malignant brain tumors can occur in combination with intraoperative mapping techniques for motor language pathways. Possibly overall survival in patients with malignant glioma tumors can also be impacted with greater extent of resection of tumors with direct visualization. So what are the fluorescence-guided surgery technologies? Well, Indocyanin Green, which is currently used for neurovascular applications, has not been used extensively for the resection of brain and spinal cord tumors at this time. There is some investigation going on, but we will not be focusing on ICG at this point. It is a nonspecific fluorophore, and it is FDA-approved for ophthalmologic indications and has been used for intraoperative angiography during cerebrovascular surgery. There are really two main technologies that are used for fluorescence-guided brain tumor resection, fluorescein sodium and 5-aminolevulinic acid. Fluorescein sodium is really a nonspecific extracellular fluorophore. It is FDA-approved also for ophthalmologic indications, such as retinal angiography, and it is used off-label for brain tumor resections. It does rely on disruption of the blood-brain barrier for distribution of brain tumors, and there is a time limit of fluorescence. 5-aminolevulinic acid, also known as 5-ALA, also known as Gliolan, as the drug marketed in Europe and Asia, is a prodrug metabolized by tumor tissue to its fluorescent form intracellularly to protoporphyrin 9. Protoporphyrin 9 is what fluoresces. It is the most extensively studied and published agent for FGS of CNS tumors throughout the world, and as mentioned, we have level one evidence supporting more complete resection of high-grade gliomas and better progression-free survival with the use of 5-ALA fluorescence-guided surgery. Here is an illustration just summarizing the fluorescence emission wavelengths. As you can see, ICG is in the near-infrared spectrum. 5-ALA and fluorescein are really within the visible light spectrum. Fluorescein emits light between 500 and 550 nanometers, and 5-ALA emits a violet-red light around 610 nanometers. Fluorescein sodium was really described back in the late 40s by Dr. G. Moore, who used it for localization of brain tumors. As mentioned, its emission range is between 500 and 550 nanometers, and fluorescein is excited by 400 nanometers of light. It's administered intravenously. Typically a dose of 3 to 20 milligrams per kilogram is given after intubation or prior to skin incision. The timing of the administration has been described, and it is not currently defined. It does rely on blood-brain barrier disruption, and when it is administered, it does travel through the cerebral vasculature, and the dura fluoresces in addition to the cerebral spinal fluid in the surrounding brain. As tumor tissue is resected, the fluorescence can leak out since it is an extracellular fluorophore. Here is some figures from Dr. Serbi's paper in Neurosurgery Focus in 2014, demonstrating the fluorescence of the tumor after fluorescein administration. You can see the red arrow pointing to the fluorescent tissue. It's interesting that if there's any part of the cortex of the brain that's contused during the opening, that will also potentially light up with fluorescein fluorescence. This is a video that Dr. Peter Nagaji from the Barrow Neurological Institute was kind to provide that I would like to demonstrate briefly the use of fluorescein for the resection of a right parietal glioblastoma tumor. You can see here the yellow tumor that's visualized under the light, and then with the white light thereafter. Fluorescein sodium and tumor types, it's mainly been described in high-grade gliomas. I do have some papers listed at the bottom for references. It's also been reported in skull-based tumors in 2010 and brain mets. There's really a paucity of literature for the use of fluorescein and CNS tumors. However, there are more papers coming out on this topic as more and more users of fluorescein describe their experience. On the other hand, 5-ALA fluorescence-guided surgery has been very well-documented globally. This is a review article, and these are images from Dr. David Roberts from Dartmouth, who really has, in North America, began the experience with 5-ALA years ago. We do have to give credit, however, to Dr. Walter Stumer from the University of Munster, who really pioneered this technology for 5-ALA fluorescence-guided surgery beginning in 1998. He has performed multiple prospective studies, including a large randomized phase three study that's provided the level one evidence we will discuss. As you can see here, from 1998, when the first patient was dosed with 5-ALA, published in neurosurgery, to 2016, we've had a surge in publications on the use of 5-ALA fluorescence-guided surgery for not only brain tumors, but now other tumor types in the CNS. The metabolism of 5-ALA really occurs through the heme metabolic pathway within the mitochondria of cells, and it does localize to tumor cells. As mentioned before, 5-ALA is a prodrug. It's administered orally. It's a sour solution that's administered and patient waits approximately three hours before being taken back for surgery. The tumor tissue fluorescence can persist 8 to 12 hours after administration, and really the agent's taken up into the blood within one hour, and protoporphyrin 9 production occurs after one hour. The profile of 5-ALA and safety are well-documented. It does readily cross the blood-brain barrier and accumulates within malignant glioma cells. As mentioned before, it's visualized after excitation of 400 to 410 nanometer light, and it is essentially non-toxic. It does cause eye and skin phototoxicity within 24 hours. Patients are kept in subdued lighting within the first 24 to 48 hours after surgery. It is metabolized by the liver, and there can be transient LFT elevation. However, all LFT elevations typically return to normal within weeks after surgery. You can see the illustration on the right just highlighting the use of the microscope emitting the blue light, which is 410 nanometers, exciting the protoporphyrin 9 that's produced by the malignant glioma cells within the tumor. So what tumors fluoresce with 5-ALA? Well, the most published tumor type are high-grade gliomas, including recurrent high-grade gliomas. There are papers now describing the use of 5-ALA for diffuse infiltrating gliomas with anaplastic foci, low-grade gliomas, appendomomas, sub-appendomomas. There are papers on brain metastases and also meningiomas. Why do tumors fluoresce with 5-ALA? Well, briefly I have an illustration here showing the metabolic pathway of 5-ALA. Really at the point of protoporphyrin production, protoporphyrin 9, the enzyme furrochelatase is felt to be lower in various tumor types, allowing for the backup and increased production of protoporphyrin 9 within the cell. And also it's felt that protoporphyrin 9 is not removed by different outflow transporters, allowing for its accumulation within the cell. We have some in vitro work that we have published this past year. Dr. Stumer has also published in vitro work with malignant glioma cells. We use human glioblastoma cells, you can see here, in comparison to human astrocytes. Human astrocytes do not fluoresce after 5-ALA administration. You can see the glioblastoma cells do very well. And then when we co-culture normal fibroblasts with breast cancer cells, you can see here that the breast cancer cells do fluoresce and the fibroblasts do not fluoresce well, again showing the selective uptake by cancer cells in production of protoporphyrin 9. Fluorescein, the Indiana group, Dr. Aaron Cohen-Gadol has shown that fluorescein is not taken up by malignant glioma cells. You can see here, after exposure up to 60 minutes, there's no evidence of fluorescence within the cells of malignant glioma cells. In a 5-ALA rodent glioblastoma model, we can see the violet-red fluorescence very well. And then when we compare 5-ALA to fluorescein, you can see the upper left-hand panel shows the fluorescent red channel of the protoporphyrin 9 fluorescence in comparison to the fluorescein where there is more diffuse fluorescence present in this rodent glioma model. This was the circumscribed tumor model shown in the prior slide. So when we describe 5-ALA fluorescence-guided surgery in patients, this is what we're visualizing under the microscope. And this is a patient I operated on which I thought I had performed a radical resection showing evidence of fluorescence present within the resection cavity that shows residual tumor. We recently published a review article with Dr. Widhelm from Austria and Dr. Wolter Stumer from the University of Münster in Germany, really going over a lot of what's discussed tonight in specific reference to 5-ALA and hybrid gliomas. I'd like to show a video here just demonstrating 5-ALA fluorescence-guided surgery in a glioblastoma patient that I performed surgery on. You can see here under white light this is a highly vascular tumor we switch on to the fluorescence mode. And you can see within the tumor bed the violet-red fluorescence of the tumor. Specific to the tumor, there's no fluorescence outside of the tumor resection cavity. This does, this permits the neurosurgeon, myself, to perform surgical resection of the tumor in real-time fashion independent of brain shift. The resection's performed until the violet-red fluorescence is removed completely, especially if it's in an area where eloquent tissue is not present. So we describe real-time localization of the tumor independent of neuro-navigation, independent of brain shift. Well, the other point that's very important to discuss is the diagnostic accuracy of 5-ALA induced fluorescence or fluorescein-induced fluorescence. Does what we see up here as tumor tissue, which is fluorescent, is very important? It's a question that neurosurgeons need to feel confident with as they perform their fluorescence-guided surgical resection of a tumor. And in the case of 5-ALA, there's no question that there's unprecedented high positive and negative predictive values for tissue fluorescence in malignant tumor presence. The specificity and sensitivity are also important. As you can see here, this is a sampling of tissue outside of the contrast-enhancing border showing evidence of tumor fluorescence past the area of the bulk tumor. This is that same patient I showed you an illustration earlier, again, showing the resection bed away from the contrast-enhancing border. Of course, there is shift here since the tumor was debulked. However, we are, you know, outside of the contrast-enhancing border by at least a centimeter in this case documenting the presence of fluorescence in likely tumor. In a review article by Dr. Vidhelm published several years ago, he was able to show with histologic comparison the evidence of fluorescence present within the tumor bulk. And as you move past the contrast-enhancing border, there's less and less fluorescence and more and more infiltrating cancer cells. So the fluorescence signal does go away as the tumor involves more infiltrating white matter tracts. This is a patient that we operated on that showed, you know, some of the results. in this area was sampled and confirmed to be tumor. This was not visualized on the MRI scan at the time of surgery. A meta-analysis was performed and published several years ago, again, confirming the high sensitivity present, in addition to high specificity rates with tissue fluorescence and malignant tumor tissue presence. These were all prospective studies that were analyzed. As you can see here in this table, I've listed many important studies that have documented the positive predictive value for the use of fluorescence-guided surgery for brain tumors. In the case of 5-ALA, these positive predictive values are in the high 90s for the majority of studies. There are two fluorescein studies that have shown high sensitivity and specificity presence for a tumor with yellow tissue fluorescence. Dr. Sumer performed an important study where he was actually able to quantify the fluorescent signal by a spectroscopy device and correlate it with histopathology. In that study, he was able to basically take tumor biopsies from tissues with strong and weak fluorescence. As you can imagine, tissues with strong fluorescence corresponded to greater spectrometric fluorescence, solidly proliferating tumor, and high tumor cell densities, while weakly fluorescent tissues had lower quantified fluorescence, infiltrating tumor, and medium tumor cell densities. Again, another study showing very high positive predictive value for malignant glioma tumor and strongly fluorescent tissue. There's been a study where patients have been administered both 5-ALA and fluorescein. This was done in Germany by Dr. Sumer's group. This study was done in four patients, two patients with glioblastoma, one with a secondary glioblastoma, and one with a recurrent low-grade glioma. The recurrent low-grade glioma did not have any evidence of fluorescence with either 5-ALA or fluorescein. The study was stopped prematurely. The surgeons felt that there was no added benefit with the use of fluorescein in the resection. As you can see here, a comparison between the fluorescein fluorescence, the lower panel CFI, and then 5-ALA fluorescence-guided surgery, BEH. We have data also supporting the use of 5-ALA fluorescence-guided surgery for recurrent glioblastomas. In a study by Nabavi et al., published in Neurosurgery in 2009, this study was a biopsy-driven study where, again, a high positive predictive value was calculated for the detection of recurrent tumor in patients who underwent chemo radiation at the time of their initial diagnosis. This was also confirmed in another study published in the journal Neuro-Oncology. The Dartmouth group, Dr. Pablo Veldez and Dr. Roberts, have been able to also quantify protoporphyrin 9 levels with contrast enhancement, fluorescence, and malignancy, again confirming the localization of protoporphyrin 9 within malignant glioma tissue that's confirmed histopathologically. There are also other indications for fluorescence-guided surgery, such as stereotactic brain tumor biopsies. This is a paper by the Vienna group where they've described the use of 5-ALA fluorescence for an intraoperative marker for tissue samples in stereotactic brain tumor biopsies. The thought is that less passes would be needed if fluorescence is detected since there's such a high positive predictive value for malignant tumor tissue with the presence of fluorescence. There's also one study with fluorescein that's described the use of fluorescein for stereotactic biopsies as well. This is a biopsy of a patient who had a primary CNS lymphoma who underwent a stereotactic biopsy of their central contrast enhancing lesion that was carrying a trigger in location. And as you will see in the biopsy needle, there is the presence of red fluorescent tissue confirming a malignant primary CNS lymphoma. Much of the difficulties associated with malignant glioma resection lie at the margin, the MRI contrast enhancing border and beyond. And we know that from multiple experience described that 5-ALA does extend past the contrast enhancing border on MRI scan. Protoporphyrin is found within the infiltrating glioma cells at the margin. We know that fluorescence is more sensitive at detecting the tumor margin than neuronavigation. And it's even felt to be more sensitive than intraoperative MRI at detecting tumor infiltration. There are studies now describing fluorescein-fluorescence-guided surgery. However, it's felt that fluorescein is kept within the confines of the contrast-enhancing border since fluorescein relies on the breakdown of the blood-brain barrier for its extracellular course into tumors. It is unclear whether fluorescein does extend past the contrast-enhancing border. This is a study by Pansiani et al. just confirming, again, biopsies within and outside the area of contrast enhancement by neuronavigation and showing the very high positive predictive values for tissue fluorescence and malignant tumor tissues past the contrast-enhancing border. In the case of 5-ALA and high-grade gliomas that lack typical contrast-enhancing features on MRI scan, there are studies that have shown 5-ALA does continue to produce protoporphyrin 9, and fluorescence is detected in these tumor types. Fluorescence is correlated with WHO grade. Obviously, malignant gliomas will fluoresce more than low-grade gliomas. And as we know, 30 percent of non-enhancing gliomas are high-grade, so there is a good chance that those tumor types may produce protoporphyrin 9 fluorescence. This is a study that was published in Neurosurgery in 2015, really looking at these different tumor imaging types that did correlate fluorescence with the higher WHO grade, in addition to the proliferative index. There is no correlation with the MGMT promoter status, IDH1 mutation status, or 1p19q deletion status with fluorescence. We have also had studies that have been done in a prospective fashion, looking at whether surgeons should perform maximal white light resection of high-grade gliomas, and then switched on the blue light afterwards to determine residual tissue fluorescence and tumor presence, as I mentioned earlier in this top panel. And the question is, is there a substantial tumor left behind? And as you can see here in Table 4, there are five prospective studies that have been performed, both the newly diagnosed as well as recurrent high-grade gliomas, where surgeons performed maximal white light resection and then turned on the fluorescence light, and the presence of tissue was found present at that time, requiring further resection. I'd like to talk about false positive fluorescence at this point, with both technologies. So as we discussed, fluorescein is a nonspecific extracellular fluorophore that really relies on the breakdown of the blood-brain barrier for CNS penetration. So when fluorescein is administered, it's seen within the dura, the CSF, and really any part of the brain that can be contused or have blood-brain barrier disruption. It can leak out during tumor resection, and really the timing of fluorescein administration is variable. Administering it too early might result in nonspecific fluorescence, and acute administration will provide detection of abnormally perfused tumor tissue. We feel that fluorescence at this time corresponds to the contrast-enhancing border present on the MRI scan. There are reports of false positive fluorescence with 5-ALAY as well. However, all the reports of false positive fluorescence are really within the immediate vicinity of viable tumor cells. There's no reports of normal brain distant from gross tumor having false positive fluorescence. There's false positive fluorescence that's been reported with recurrent gliomas who've undergone prior adjuvant therapies. The thought may be reactive astrocytes may play a role. The group in Japan, Atsuki et al., has described this in 2007. Fluorescence can occur with radiation necrosis as well. False negative fluorescence, we know that high-grade gliomas infiltrate normal brain. So as you move past the tumor bulk and into the infiltrating tumor zone, the fluorescence signal is lost. So that could be a reason to have no fluorescence present when tumor is present. We do have technologies that have been described by multiple groups, specifically spectroscopy and confocal microscopy, where protoporphyrin 9 can be visualized when the microscope cannot visualize the protoporphyrin 9 fluorescence. Photobleaching may be a cause for false negative fluorescence. And really the timing of 5-ALA administration is important. It's orally administered. The patient is taken back to surgery within 3 hours after administration. Taking the patient too early to surgery may be a problem. However, fluorescence can be seen up to 12 hours after administration. As mentioned before, we do have Level 1 evidence for 5-ALA fluorescence-guided surgery leading to more complete resections and better progression-free survival. You can see this was done in a study by Walter Stumer, published in Lancet Oncology in 2006. A follow-up study was performed in the Journal of Neurosurgery in 2010. And you can see on the left panel that the more complete resection of gliomas was performed, high-grade gliomas was performed in the patient group that received 5-ALA in comparison to the group that underwent white light resection. There also was a significant increase in PFS6 from 21.8% to 35.2%. This study was not powered for overall survival. And furthermore, there were differences in adjuvant therapy management of patients after they underwent their surgical resection. One caveat of this study too is that many patients did not undergo image-guided resection with neuronavigation in this trial. We have a number of other studies that have shown greater extent of resection of tumor after 5-ALA resection. This involves tumors that are newly diagnosed high-grade gliomas in addition to recurrent. And as you can see here that the resection rates are increased with 5-ALA. We have completed a study in the U.S. also confirming this with a greater extent of resection in patients in comparison to a control cohort. We published this initial result in 2014 when 16 patients were resected with 5-ALA in comparison to 37 control patients. In this Phase 2 study, 72 patients were completed. Another interim publication described the greater extent of resection of these patients. In this study here, 30 patients were dosed with 5-ALA and had better overall survival and more complete resection of their high-grade glioma. Fluorescence safety is very important when we discuss fluorescence-guided surgery. Fortunately, we have two agents that are very safe for patients. In higher concentration, fluorescein has been known to cause anaphylaxis. It's unclear how many patients have been administered fluorescein for neurosurgical applications. We know that thousands of patients have undergone retinal angiography and angioscopy for decades with fluorescein. We know that with 5-ALA, probably close to 60,000 patients worldwide have undergone 5-ALA fluorescence-guided surgery. It was approved in Europe in 2007. There's no deaths reported after 5-ALA. There was really one reported overdose of 5-ALA in a patient who developed respiratory depression. Common side effects were headaches, nausea, and skin sensitivity. A small portion of patients can have elevated LFTs, however, all return normal by 6 weeks. Neurologic deficits after fluorescence-guided surgery have been reported in the randomized phase 3 study with 5-ALA. There were more deficits found in patients that underwent 5-ALA fluorescence-guided surgery within 6 weeks. However, at 6 weeks post-op, there was no significant difference between the 5-ALA group and the white light treated group. Some other tumor types that have been described for fluorescence-guided surgery include spinal cord tumors. This was a publication by the Vienna Group led by Dr. George Vinhelm. I also have a video here just showing an intramedullary spinal cord ependymoma showing avid violet-red fluorescence within the cord with fluorescence visualization. 5-ALA has also been described in meningiomas. This is a new area that's recently had several publications documenting the experience with 5-ALA fluorescence-guided surgery. This is another video kindly provided by the Vienna Group showing a CP angle meningioma and fluorescence present. In the pediatric tumor population, there have been case reports. The first case report was actually from the United States by Dr. Ruge, who described the use of 5-ALA in a pleomorphic xanthoastrocytoma. There have been other case reports described as well. There's no prospective study, however, at this point documenting the use of fluorescence-guided surgery in pediatric tumors. A recent European survey was collected on 78 pediatric patients at different centers who underwent 5-ALA fluorescence-guided surgery. They were able to describe a consistent experience with high-grade gliomas, where over 85 percent had tumor fluorescence present. The majority of ependymomas fluoresced after 5-ALA administration. However, fluorescence was not found useful in PNETs, gangliogliomas, meduloblastomas were only a quarter fluoresced after 5-ALA administration, and as you would imagine, WHO grade 1 pilocytic astrocytomas. There's no fluorescein fluorescence-guided surgery in pediatric tumors reported to my knowledge at this time. 5-ALA has also been used to detect anaplastic foci and presumed low-grade gliomas. As we discussed before, we know that patients who have non-significant contrast enhancement may have evidence of a malignant glioma despite the appearance of a low-grade glioma. We feel that maybe 40 to 50 percent of patients may have evidence of an anaplastic glioma despite the imaging. So, PET and intraoperative fluorescence can detect these foci, and 5-ALA has also been used in this patient population to localize these foci and perform resection and adequate sampling for proper treatment of these tumors. This is an example from that paper just showing the imaging features of a glioma that appears to be possibly a low-grade glioma with non-significant contrast enhancement, hyperintensity with two-weighted imaging, but had evidence of anaplastic foci present at the time of surgery. You can see here the same type of patient where there's no significant contrast enhancement, and then fluorescence present within and during the tumor resection that's confirmed by histopathology. So where do we go from here? Well, you know, we can reliably visualize the tumor bulk and margin with fluorescence-guided surgery to prevent more complete resections. Fluorescence-guided surgery provides real-time image guidance independent of neuronavigation and brain shift. Can we extend our resection past the tumor-enhancing border? You know, I think we do have evidence for that. Can we use this for low-grade gliomas? I think, you know, we're still trying to understand the role of fluorescence-guided surgery in low-grade gliomas. And can we combine this with other imaging modalities to help with our surgery? Well, this is just one example from work we did just using a handheld device that could detect fluorescence at a much greater sensitivity than the operative microscope. So this is a microscope, and in the study we were able to compare the use of the handheld device to the microscope, really showing three to four orders of magnitude greater detection sensitivity of cellular tumor fluorescence after 5-AOA administration and protoporphyrin 9 production. There's other examples of these types of technologies that are being developed to complement the operative fluorescence-fitted microscope. Imaging is also a big player when it comes to our ability to resect tumors. We know that with high-grade gliomas, they infiltrate into the normal brain centimeters away from the main tumor mass, and we have the ability to visualize these tumor cells with metabolic imaging of the brain, specifically magnetic resonance spectroscopic imaging, where we can now look at metabolites such as choline and NAA metabolites. And the ratio of choline over NAA can allow us to sensitively detect tumors outside of the contrast-enhancing border in addition to the T2-weighted image. This is an example of documenting this and providing further evidence of the infiltrative nature of these gliomas where the contrast enhancer really does not show this contralateral involvement of the tumor. We can combine this type of imaging with fluorescence-guided surgery to maximize our ability to resect high-grade gliomas in this study, which was recently published this year, documenting the use of neuronavigation with magnetic resonance spectroscopic imaging in combination with 5-alay fluorescence-guided surgery that was confirmed by histopathology examination of tumor. Fluorescein fluorescence-guided surgery in the United States is really used off-label for high-grade glioma resections and requires IRB approval and patient consent through a protocol. 5-alay fluorescence-guided surgery in the U.S. has been approved in Europe since 2007 and registered in 34 countries outside of the U.S. Here in the U.S., we can only use 5-alay through an IND currently. We approached the FDA in 2011, and the European experience was presented at that time, in addition to thousands of patients, safety data, and the PFS6 improvement. At that time, the FDA requested clinical benefit discussion with patients in receiving 5-alay. We have since undergone the training of many neurosurgeons on the use of 5-alay within the United States. Currently, it can only be used with an IND. It has, however, received orphan drug approval in 2013 by the FDA and has been given a go-ahead for a new drug application with a fast-track submission, which will occur later this month in early October. The proposed FDA indication and label will be that glialin, which is 5-alay, is an imaging agent to facilitate the real-time detection and visualization of malignant tissue during glioma surgery. So is 5-alay ready for prime time? Well, I think there is a lot of experience with 5-alay that can provide visualization of the tumor and the margin. It provides real-time guidance and can extend the resection of tumors past the contrast-enhancing border. The agent is safe. Thousands of patients have been dosed, multiple tumor types for us, and it's improved in 34 countries. This will be the completion of the webinar. I would like to acknowledge the ONNS once again and the Neurosurgery Research and Education Fund. Also, Leica for allowing us to provide this webinar complementary this evening. I'd like to acknowledge NX Development Corporation, who is really leading the way with the NDA to the FDA for 5-alay. Photonomic is the company in Germany that provides the agent at this time, Dr. Anne Morin Ulrich Kochiesa. And of course, Drs. Walter Stumer, Peter Nakaji, and George Witthelm. I'd like to acknowledge their help with this webinar. This is my email. If you have any questions, I would like to go ahead and go through the questions that were provided here. Okay. So, it looks like everyone can hear us well. What is the protocol for administration of 5-alay for glioma surgery, dose, and timing? So, 5-alay has to be given approximately three hours before the patient is taken to surgery. So, typically, you know, if you're going to do the cases, the first case, you have to wait a couple hours. So, that's why sometimes it makes sense to have a short case first, dose the patient, do your short case first, and then move to the 5-alay fluorescence guided surgery. Now, some centers are able to administer the dose of 5-alay in the early morning. That has been documented. In that case, you could start with the first morning start with administration early. Okay. There is also evidence of optical coherence tomography being used to operate on brain tumors. Is 5-alay better than OCT in terms of surgical accuracy for resection? Well, I think, you know, there are various technologies being examined now. Raman spectroscopy is another one which is being referred here. I think, you know, we're studying each of these technologies and their limits. I think certainly, you know, it's important to understand, you know, what role each technology will play. But fortunately, 5-alay is very good at providing tissue tumor fluorescence and can reliably provide fluorescence for the real-time visualization of tumor. Is NX Development Group the only company that offers 5-alay in the USA? If not, where else can you get it? Currently, NX Development is the only company that's able to procure the drug from Germany. And it is provided through an IND. So, if you have any questions, please refer your questions to NX Development Corporation. My question is, my microscope is OPMI PICO. Can I do fluorescence guided surgery using this microscope? I'm not quite sure how to answer this OPMI PICO. I don't know. There are, you know, ZEISS, Leica both have the ability to perform fluorescence guided surgery. There are other microscopes as well in Europe and Asia that have this ability as well. So, I don't know if I was able to answer that question. We discussed the role of pediatric tumors. I think, you know, there is a lot to learn from the pediatric population. There is a paucity of literature. We really only have that European survey that described neurosurgeons' experience with different tumor types in the PEDS population. But this is certainly an area that deserves much more investigation. Another question, if fluorescence is detected with neuronavigation outside of the delineated area in the preoperative MRI and intraoperative MRI performed and the fluorescence again is detected again outside of the target planned area, what should be done with the lesion if it is definitely in an eloquent area? Well, this is an excellent question. So, obviously, you know, we don't advise neurosurgeons to take out everything that fluoresces, especially if it's in an eloquent area. So, that's why we depend on other technologies such as cortical subcortical mapping to define motor pathways during resection. So, I think in this situation, you know, this is the surgeon's discretion, especially in an area of eloquence. You know, I think if fluorescence is detected even after an intraoperative MRI with 5LA especially past the contrast enhancing border, that likely represents tumor. So, the question is, is it safe to resect that tumor? And that really has to be the decision of the neurosurgeon at the time of surgery and possibly using, you know, other types of intraoperative techniques to determine how far the resection should be extended. What is the ideal time of 5-AOA administration prior to surgery? I think, like, as we mentioned, up to three hours. In Europe, actually, the label says two to four hours, but we've kept it three hours in the United States. Is there a difference in fluorescence intensity of tumors based on location within brain parenchyma? That's an interesting question. I don't think there is. You know, I think the fluorescence of tumor tissue shouldn't change with the location of the tumor. It's really based on the tumor grade that really affects fluorescence, as we discussed during the webinar. The higher the tumor grade, especially malignant glioma and glioblastoma tumor types, avidly fluoresce. I do think contrast enhancement is important, and the more contrast enhancement present within a high-grade glioma, the better the fluorescence is present. Okay, how would one practically know intraoperatively that one has resected the tumor and the further fluorescence seen in that from contused brain, since it is not practical to do MRS every now and then? This is a very important question, and I think with 5-ALA, we don't see this phenomena where the brain is contused and there's fluorescence that appears. This is mainly an issue with fluorescein fluorescence guided surgery due to its blood-brain barrier disruption requirement. So I think that's really the situation where it's found. What are the handheld devices used for greater signal in the session than the microscope called? Well, you know, there's various, you know, types of technologies being developed. There are handheld confocal devices, there are handheld spectroscopy devices that are being developed. So I don't think we have one that's being marketed quite yet, but there will be some in the future that will complement the microscope. How can we improve neurologic function, which is lost during the course of the disease? Will 5-ALA help in any way? Well, I think this is a good question, too. So I think, in my experience, when I've used 5-ALA for fluorescence guided surgery in combination with mapping techniques, I've really found that to be very powerful, permitting me to push the resection of the tumor, knowing where the approximate location of the eloquent pathways is in relationship to the fluorescent tissue tumor regions. And I think that's really helpful to be able to selectively resect the tumor while minimizing any damaged eloquent pathways. Any difference in uptake between solid versus cystic lesions? Any study on whether tissue beyond fluorescence margin with positive pathology or presence of tumor? So actually, necrotic regions of hygrid gliomas do not fluoresce well, so if that's what you mean. The presence of fluorescence past the tumor contrast enhancing margin is definitely present with 5-ALA, and we've shown that with biopsying of tumors, confirming histopathologic presence of tumor past the contrast enhancing border, even with real-time intraoperative MRI, to take into account brain shift. So cost, I think, is an important discussion. I think the cost, you know, is yet to be seen for the United States. It's not FDA approved at this time. In Europe, it's about 1,000 euros for a vial of 5-ALA, so I think, you know, I think that will be something that will be determined at a future time. Thank you for excellent question. As regard to sensitivity and specificity, do you recommend its routine use in a meningioma and apendymoma cases? One of the questions that I have, I think, I think we all know that we can resect, you know, a low-bar meningioma well, but I think at the skull base, being able to visualize the hyperostatic bone involved by meningioma would be important, and that's been shown with 5-ALA fluorescence guided surgery that the involved surrounding bone could fluoresce and guide removal. What else is necessary besides the microscope for fluorescence guided surgery? Well, you have to have the drug, too. So once you have the, you administer the drug and your microscope is fitted for fluorescence, you're almost ready to go. You do need to be trained, though. That's a very important point that any neurosurgeon who's considering fluorescence guided surgery really needs proper training, going to a formal training. We've provided training in the United States. The US has a risk management program for training in addition to Asia and Australia, and I think it's important to be certified prior to moving forward. Once 5-ALA is approved and available in the US, do you think it will still, do you think there will still be a role for fluorescein use? My opinion is no. I don't think there'll be a role for fluorescein once the FDA approves 5-ALA. I just feel that it's a much more sensitive agent. It's not a nonspecific fluorophore. It's metabolized within the tumor cell to its fluorescent form, and, you know, I really think it's a wonderful agent that can be used safely in patients. Do you use this for all of your tumors now, regardless of what your differential diagnosis is? I actually, because I use it under an investigational new drug, IND, I do have to use it for tumors I believe are high-grade gliomas. However, in Europe and Asia, in Australia, you can use it, since it's approved now, for other tumor types, and I showed you some videos of spinal cord tumors, and again, the meningiomas. What is the difference in cost between fluorescein and 5-ALA? A good cost. Fluorescein, as you know, is an inexpensive agent. The cost of 5-ALA has yet to be determined in this country, and, as I mentioned, in Europe, I told you it's approximately 1,000 euro per vial. I don't know the cost of it in Asia and Australia. How does a sprout join your multicenter trial? Okay, well, I think, you know, if you guys can email me, my email is here. I will provide more information on the multicenter trial that we have open now in the United States. This is a trial that's really a six-week trial that is looking at histopathology and sampling of fluorescent tumor tissue regions after 5-ALA administration and high-grade gliomas. I'm not sure my microscope has a blue light. How do I find the setting? If it's not available on the microscope, how do I get it, please? Well, I would recommend that you speak to your microscope representative, your Leica or Zeiss representative. They should be able to tell you if your microscope is set up for fluorescence. If it's not, don't fret. Microscopes can be fitted with the fluorescence module without having to get a new microscope. Okay, at one point, you mentioned that tumor necrosis may also fluoresce a 5-ALA, if I heard correctly. How do you deal with this phenomenon during surgery? Well, I think, you know, we know that tumor necrosis and treatment effect always has tumor present as well. So I think, you know, if there's evidence of tumor necrosis, there's going to be evidence of tumor as well in most cases. So I think, you know, the pathologist will help with the frozen diagnosis, but I think debulking the tumor typically is still required at this point because you're still taking the patient for debulking and relief of mass effect. What is the real impact of 5-ALA and GBM recurrence and survival? Well, we know that in GBM recurrence that tissue fluoresces despite patients undergoing chemo radiation. It's hard to say at this point whether there's an increase in overall survival. I don't think the literature has shown that yet. The level one evidence that we have is for newly diagnosed high-grade gliomas. Can training be obtained in the U.S.? Well, at this point, we're organizing another training. Stay tuned for that. Please contact myself, and I will be sure to let you know. Apart from tumors, can this technology be used to visualize nerves as eggs in the foramen, thinking about expanded uses in spines? Well, that's an interesting question. I don't think so. I don't know why nerves would metabolize 5-ALA or fluoresce with fluorescein, so I don't think there's an indication for that. Is there any evidence that there is a better survival using this technique? Well, there are actually some studies that show better overall survival with fluorescence-guided surgery in high-grade gliomas, so there are studies that have confirmed this. Are there any interassociations between 5-ALA and brain cellin? I don't know what that means. Oh, brain swollen. Okay, maybe you mean cerebral edema? No, there isn't. The only thing that we described is in the resection cavity, could there be reactive astrocytes that can fluoresce? There is evidence that fluorescein-fluorescence-guided surgery can be, I'm sorry, fluorescein fluorescence can be present in cerebral edema, and that's been published documenting this. Are there other fluorescence agents in neurosurgery being developed? Yes, there are some other types of targeted type fluorophores that are being developed and are being considered for use in more expanded patient trials. Is there any work on chordomas and 5-ALA? To my knowledge, I don't know. I don't think so, but there may be a case report on this, but that's an important question. Once FDA approved, will 5-ALA be used in all patients with brain tumors? Well, once 5-ALA is approved, it will be standard of care to administer 5-ALA to patients with suspected high-grade gliomas. And even patients with non-enhancing gliomas that may represent high-grade gliomas. What about the utility of 5-ALA in pituitary adenomas and residual adenomas? There are papers documenting the efficacy of 5-ALA in pituitary adenomas. However, there is some controversy on this and whether pituitary adenomas fluoresce uniformly after 5-ALA is administered. So I think that's something that needs to be studied further. Also, with the use of better handheld technologies, especially with endoscopic approaches, that question may be answered well. Does the company that produced the drug have resources available to help with the IND process? Well, the answer to that is yes, but I think they would prefer to have you take part in the multicenter trial that we have for high-grade gliomas under my existing IND. I think at this point, you know, setting up another IND just takes, is just a long process. Furthermore, there will be other studies being done, hopefully in the near future, for pediatric tumors, for stereotactic brain biopsies, for mini-gliomas, and also for possibly pituitary tumors. Does 5-ALA fluoresce and highlight cortical dysplasia and therefore be helpful in epilepsy surgery? I don't know the answer to that question, so I would purely be speculating. Can you expand on the utility of this in the context of radiation necrosis versus recurrent tumor? I think we answered this question before. Is frozen section during surgery set to check tumor margins if 5-ALA technology is being used, or is it not required then? You know, that's a good question. I think I don't think you need to send off that tissue at the margin that fluoresces for a frozen. I think based on the large experience by others that has been published now, that if you see tissue fluorescence, that that does highly predict tumor. Can you use this for stereotactic biopsy for deep-seated small tumors? Yes, definitely. And I think I showed a video for a CNS lymphoma, but that also applies to high-grade gliomas as well. Can you use it for brain secondaries? I think you could also use that as well for metastases. And then, you know, the concept of whether, you know, using fluorescence-guided surgery will lessen the chance for a near-term reoperation. Patients who've had kind of an open biopsy that then require a formal, more formal debulking a month later, fluorescence-guided surgery, I think, does help with minimizing those types of situations. How much does this technology cost? I think we talked about that before. Okay. I think we've reached the end of the questions. Again, I just want to thank everyone for joining our webinar tonight, and I want to thank Katie and Renan for their help. And please reach out to me by email if you have any further questions. Thank you very much.

Fluorescence-Guided Surgery

5-aminolevulinic acid

Fluorescein sodium

Brain tumors

Protoporphyrin 9

Diagnostic accuracy

Tumor resection

Spinal cord tumors

Meningiomas

Ongoing research