Welcome to the webinar, What is the Surgical Benefit of Utilizing 5 ALA Sphophorescent Guided Surgery of Brain Tumors? You'll be hearing a presentation from Dr. Haja Panias on this very timely subject. But before we get started, I want to take a few minutes of your time. If you wish to ask a question during the presentation, please use the chat feature located in the lower left-hand corner of your screen. At the end of the presentation, there will be a 10-minute question-and-answer session. As a part of NREF's efforts to provide you with timely topics and interesting speakers, we'd appreciate it if you would fill out the evaluation at the end of the webinar. Now, moving along to our session, please welcome Dr. Haja Panias, who will be speaking to us on fluorescence guided surgery. Thank you, Katie. Welcome everybody. Thank you for taking the time out to participate in this webinar. I will be directing tonight on 5 ALA fluorescence guided surgery for malignant gliomas. I would like to thank the American Association of Neurological Surgeons. Before we start the presentation, though, I do want to show my disclosures. And I do want to remind the audience that 5 ALA, also known as Gliolan, is an investigational drug and is not currently FDA approved in the United States. It is approved in other countries around the world, and we will be discussing this under the investigational new drug 1.1.2.2.4.6. I do want to acknowledge the Neurosurgery Research and Education Foundation and LICA for allowing us to have this webinar tonight, and again, the AANS for organizing this. Our agenda for the webinar tonight will be an introduction to fluorescence guided surgery, understanding the metabolism of 5 ALA, and looking into some experiments involving 5 ALA in vitro and a rhodochglioma model, understanding the sensitivity and positive predictive value of 5 ALA fluorescence guidance, 5 ALA tissue fluorescence in malignant gliomas, extent of resection, and a phase 2 study interim results. We will discuss anaplastic foci and diffuse infiltrating gliomas in fluorescence guided surgery, safety with 5 ALA, and the regulatory state of 5 ALA in the United States. Fluorescence guided surgery for brain tumor has been around for over 10 years. It's actually a topic that's timely now as more and more surgeons are becoming familiar with this technology. Summary, this really provides real-time intraoperative visualization of malignant brain tissue, guides the neurosurgeon during their surgery for malignant brain tumors. Because of the real-time visualization of brain tumors with fluorescence guidance, surgeons are able to perform more extensive resection of tumors, and especially with tumors with infiltrative biology, the neurosurgeon is able to visualize the tumor margin in a fashion. Fluorescence guided surgery may permit safer resection of tumors in combination with other intraoperative mapping techniques, and may impact overall survival of patients with malignant gliomas. In the past, three main technologies have been used for fluorescence guided brain tumor resection. Endocyanin green, which is a nonspecific fluorophore, FDA approved for ophthalmologic indications has been used in the past. It's really a near-infrared imaging fluorophore that's been used, but really hasn't been used a lot in brain tumors due to the low fluorescence and need for NIR imaging. Fluorescein sodium has been used, especially more recently, by various groups. It is also a nonspecific fluorophore. It is FDA approved for ophthalmologic indications, just like endocyanin green. It does rely on disruption of the blood-brain barrier, and accumulates in tumors due to disruption of the blood-brain barrier. And there is a time limit of fluorescence, typically of two to three hours after intravenous injection. There has been some safety issue reports with anaphylaxis, but it can be used for malignant gliomas. Tonight we will be discussing 5-Aminolevulinic acid. It's a pro-drug. It's actually metabolized by tumor tissue to its fluorescent form, protoporphyrin 9, which we will be discussing. I do need to acknowledge Dr. Walter Stumer from the University of Munster in Germany, who really has pioneered this technology for fluorescence-guided surgery beginning in 1998. He has completed numerous studies, including a randomized Phase III study. This slide really shows you the concept of tumor fluorescence and fluorescence-guided surgery. This was provided by Dr. David Roberts, who really was the first to perform 5-ALA fluorescence-guided surgery in North America at Dartmouth. You can see on the left-hand side, with standard white light, in the resection cavity, subcortical white matter. Then when the blue filter is turned on, you can see the island of violet-red fluorescent tissue signifying tumor. This was published in a manuscript in 2010, where this figure was cited. The metabolism of 5-ALA really relies on the mitochondrial heme biosynthesis pathway. Patients will drink 50 mLs of the 5-aminolevinic acid that's been mixed by the pharmacist with a dosing of 20 milligrams per kilogram. The patient drinks this two to five hours prior to surgery. The 5-ALA is rapidly taken up into the blood from the GI system within one hour of oral administration. We typically wait at least three hours before surgery, two to five hours in Europe, it's actually two to four hours, our IND in clinical trial in the United States, we wait three to five hours. More importantly, however, tumor tissue fluorescence can persist for eight to 12 hours after a single oral administration. 5-ALA is not re-dosed, it's given in a one-time fashion. As you can see here on the lower left-hand portion of the PowerPoint slide, the 5-ALA is taken up by the glioma cell and into the mitochondria where the 5-ALA is metabolized to protoporphyrin 9. Protoporphyrin 9 is the fluorescent metabolite of 5-ALA and is what is visualized intracellularly in tumors. This is the main difference from the other fluorophores we discussed which are extracellular. 5-ALA is taken up by the cancer cells, metabolized to its fluorescent form, protoporphyrin 9. Next step in the metabolic pathway is formation of hemoglobin with the addition of heme. Here we're able to appreciate the specificity of 5-ALA for tumor cells. You can see here normal human astrocytes on the left, glioblastoma cells that are EGFR B3 overexpressing on the right that have been treated with 5-ALA. You can see that the normal human astrocytes do not take up the 5-ALA and metabolize it to protoporphyrin 9. However, the glioblastoma cells in the lower right-hand side do show avid fluorescence intracellularly once again denoting the production of protoporphyrin 9. In another experiment mixing breast cancer cells with fibroblasts, we were able to administer 5-ALA and really visualize the breast cancer cells nicely due to the production of protoporphyrin 9 and violet-red fluorescence. The fibroblasts, which are normal, do not produce the protoporphyrin 9. Again, this is intracellular fluorescence, which is used in brain tumor surgery. When we look at a rodent GBM model, and this is data that's currently pending publication, you can see here in a xenograft model in a rodent glioblastoma in the upper left-hand corner of the slide A. On slide B, you can see with blue light excitation, the protoporphyrin 9 is very avidly seen in the red fluorescence decoded in the lower hand side of the slide from the MRI tumor, including pathology in portion D. The excitation of protoporphyrin 9 occurs in the 400 nanometer wavelength range and emits in the 600 nanometer range. This slide is interesting as it is able to compare 5-ALA with fluorescein. The upper left-hand portion of the slide shows 5-ALA and fluorescein on the lower left-hand side. Because of the intracellular end taken by the tumor, we're able to see a more circumscribed fluorescence involving the xenograft in comparison to the fluorescein administration where there's fluorescein outside of the tumor in areas where the blood-brain barrier is broken down with no presence. So why do tumors fluoresce? Well, there's several proposed mechanisms. One of them includes decreased ferrochelatase activity permitting the accumulation of protoporphyrin 9. You can see that here in this biosynthesis pathway where the ferrochelatase is really allowing the formation of heme with the addition of iron. Other mechanisms of tumor fluorescence include increased 5-ALA uptake by the tumor cells or disturbance and outflow of protoporphyrin 9. There's some ABCG2 receptors that have been implicated with the ability for tumors to fluoresce as well. The 5-ALA profile, it is a heme precursor. It actually exists in the normal body and it is part of the heme biosynthesis pathway that we discussed. When we administer it orally, we're exogenously overloading the system and in glioma cells we discussed why that protoporphyrin 9 can accumulate. The ability to visualize protoporphyrin 9 is needed with a microscope that is able to emit a blue light and excite the fluorophore intracellular protoporphyrin 9, blue light that's emitted from the modified microscope is 410 nanometers. Again, the visualization of the fluorescence is in the 600 nanometer wavelength. 5-ALA is essentially non-toxic. It is, however, metabolized by epithelial tissues such as the skin, so patients can develop skin sensitivity. After being administered 5-ALA, we keep patients in subdued light conditions after their craniotomy and surgery. 5-ALA is metabolized by the liver and there are elevations in liver function tests in patients that occur and they do come back to normal after several days to weeks. Here is a nice figure, a nice slide showing the concept of fluorescence-guided surgery. This is a glioblastoma case where maximal white light resection was performed on the left side and then illumination with blue light allowed for visualization of the tumor visual margin. A lot of our talk and webinar tonight is based on a publication that was just published in the Neurosurgery Journal. This month, November 2015, I do want to acknowledge the co-authors of that study, Dr. George Vidhelm at the University of Vienna, and of course Dr. Walter Schumer, who we've already acknowledged who's at the Department of Neurosurgery as chairman at the University of Munster in Munster. Please refer to this review article if you have any further questions and I'll be happy to answer by email if there are other questions. And once again, at the end of this presentation, we will be going over all the questions you have, so please post any of your questions. Don't be afraid to ask anything. I would like to show a video for the fluorescence guided surgery to understand the concept in real time. So this is a glioblastoma case where white light resection is being performed. There's some blood in the resection cavity. We will now be switching over to the blue light mode with the switch and immediately we're able to see the excitation of the protoporphyrin 9 within the malignant glioma tissue. You can see this in the violet red. And this allows the neurosurgeon to dissect the tumor based on the real time visualization of the violet red tissue. And again, one of the benefits which we will discuss is the lack of reliance on neuronavigation at this point since we can directly visualize the tumor and the visualization of the tumor margin as we move out of the gray resection of the tumor. Violet red does become pink at the margin. You can see here there is some lighter pink color as the more violet pink tumor is resected. And that tells us that we're moving out towards the tumor margin into the areas of infiltration of the tumor. So 5ALA fluorescence guided surgery really provides real time localization of the malignant tumor. It is independent of neuronavigation. It is independent of brain shift. And these are three very important concepts that really support the use of fluorescence guided surgery during resection of malignant brain tumors. Another important concept which we will spend some time now during the webinar is really understanding the diagnostic accuracy of 5ALA fluorescence guided surgery. As a neurosurgeon resecting tumors, we want to feel confident with our ability to dissect the tissue that is truly malignant glioma tissue. So one of the questions that's important to understand is does 5ALA induced fluorescence mean malignant glioma tissue? The answer to that question is 5ALA demonstrates unprecedented high positive and negative predictive values, specificity, sensitivity for delineating tumor. Now I don't want you to take my word for it and I think we now can demonstrate some evidence to support those statements. So this is a nice article published by my colleague in Vienna, Dr. George Bidhelm, where this concept of malignant tissue and fluorescence and histopathology is correlated. And as you can see in the upper portion of the figure, in the centrally necrotic portion of the glioblastoma tumor, we don't see much fluorescence. Now it turns out there is fluorescence present in the necrotic region of the tumor. However, because of the microscope inability to visualize this lower amount of fluorescence, it does not appear fluorescent. Now as we move into the tumor bulk in 2A, you can see that there is strong fluorescence and that correlates with compact tumor. And then moving to row 3A, B, C, and D, we can see that at the tumor periphery the fluorescence is vague, it's more pink. Again, that shows infiltrating tissue. We know that the 5ALA tumor fluorescence extends past the contrast-enhancing border. That has been shown by a number of investigators and we're able to see pink and less strong fluorescence in the infiltrating tumor margin. This is an example of a case that I performed where we're at the tumor margin with neuronavigation in the upper right-hand side sampling tissue to really understand the specificity, sensitivity, positive predictive value of 5ALA tissue fluorescence correlating this with histopathology. As you can see, samples from this region do show strong fluorescence. And again, the question is, is this tumor in this region? And there for sure is some brain shift that's associated with the craniotomy, but even with this brain shift, we're outside this area of contrast enhancement. The question arises, is this tumor? And with fluorescence-guided surgery, we can say confidently that this area of pink tissue fluorescence is associated with tumor. A recent meta-analysis published in PLOS One in 2013 really did a nice job analyzing the sensitivity and specificity of various studies that have been published effectively on 5ALA fluorescence-guided surgery for glioblastoma. As you can see here, the sensitivity and specificity numbers are very high here with 5ALA fluorescence-guided surgery in GBM. When we discuss the positive predictive value, we really want to understand what is truly positive with 5ALA tissue fluorescence and correlates with malignant histopathology. And I think that is a very important point as we discuss the strength of 5ALA fluorescence-guided surgery and the ability of neurosurgeons to use this confidently to resect malignant glioma tissue. As you can see in this slide here, the positive predictive values are all in the 90s, except for one study, which was 89%. And actually, most of these studies are above 95% with positive predictive value correlating 5ALA tissue fluorescence with malignant glioma histopathology. It's unprecedented. Some of the earlier studies that really hammered this out were by Dr. Walter Stumer. In 2000, he was able to document biopsy fluorescence and really show that the positive predictive value of malignant tumor histopathology and tissue fluorescence was close to 99%. Fluorescence visualization was associated with tumor in almost every case. A more recent study by Dr. Stumer's group actually went further and was able to define and quantify fluorescence with spectrometric fluorescence and correlate that with histopathology. In this study, they were able to really show that strong fluorescence correlated with higher tumor cell densities, and weak fluorescence corresponded to lower spectrometric fluorescence infiltrating tumor and medium cell densities. So the study was really able to quantify fluorescence with spectrometry, was able to understand histopathology and correlate it with intraoperative imaging. In this study, tumor biopsies were taken from tissues from strong and weak fluorescence areas, and again, to reiterate, the strong fluorescence corresponded to greater spectrometric fluorescence, solidly proliferating tumor, and high tumor cell densities. Weak fluorescence corresponded to lower spectrometric fluorescence, infiltrated tumor, and medium tumor cell densities. And in the study, the positive predictive value in strongly fluorescent tissue was 100% for malignant gliomas, 95% for weakly fluorescing tissue, published last year in Neurosurgery. We know also that 5-ALA fluorescence-guided surgery is very effective and sensitive, also with high positive predictive values in recurrent glioblastoma cases. In this Phase II study by Nabavi et al. in Neurosurgery in 2009, 36 glioblastoma recurrent patients were analyzed in this multi-center perspective study. Biopsies were taken from strong and weak fluorescent areas at the margins after finishing a maximal white light resection. These margins were biopsied, and they appeared either normal or pathologic under white light. A total of 354 biopsies were taken in this study. In the areas with strong fluorescence, there was a positive predictive value of 98% for areas of weaker fluorescence, where there was 181 biopsies taken, the positive predictive value was 95%. Finding both the strong and weak fluorescent tissue areas, the positive predictive value was 97% for those areas. This table just summarizes those findings. Summary, 5-ALA for recurrent gliomas is highly predictive. Dr. Roberts, in 2011, was able to thoroughly analyze biopsies taken from 11 glioblastoma patients who were newly diagnosed, 124 biopsies were taken, 86 biopsies taken from fluorescing areas, and 38 were taken from non-fluorescing adjacent areas. Those that were fluorescing after 5-ALA administration, 86 of those biopsies, 82 had evidence of histopathologic malignant glioma correlating with a positive predictive value of 95%. Those biopsies where no fluorescence was visualized, 38 in total, there were 28 biopsies with tumor and those samples, negative predictive value was 26%. In adjacent areas, tumor cells are found with high probability even when there is no visible fluorescence, published in the Journal of Neurosurgery, 2011. In a study by Dr. Dave Roberts' group by Pablo Valdez et al. in 2011, this study was a neat study, actually quantifying the protoporphyrin 9 concentration in tumor tissues, also correlating imaging and microvascular proliferation in pathology, and this was able to correlate with tissue in the upper portion of the figure. The malignant glioma tumor margin is a challenging area during resection of these high-grade gliomas, and this is an area where the tumor tissue can be difficult to visualize even with a microscope and white light. We believe 5-ALA-induced tumor fluorescence is very helpful at the tumor margin, and as we have discussed, extends past the area of contrast enhancement MRI scan. This is done by multiple studies in groups, the two references below, but protoporphyrin is definitely found within infiltrating glioma cells at the margin, and fluorescence is more sensitive at detecting the tumor margin in neuronavigation. Based on some of the main points we discussed, the difficulty with neuronavigation, of course, is brain shift after craniotomy and tumor resection, especially when you get to the portion of surgery at the tumor margin. When comparing 5-ALA to intraoperative MRI, in a recent study by Koberger et al., there was a higher detection rate for tumor infiltration at the tumor margin with 5-ALA-induced fluorescence in comparison to intraoperative MRI. One concept that's important to note here is tissue fluorescence after maximal white light microsurgical resection. Even though we feel that we may have gotten the majority of the tumor, the question that arises, is there substantial tumor left behind? An example that I show here, where a maximal resection had been attempted, and when the blue light was turned on, there was a fair amount of tumor tissue fluorescence remaining that required further resection. There have been five prospective studies examining this method of surgery, where maximal white light conventional resection was performed, examining the presence of residual tissue fluorescence. In these five studies, there was visible fluorescence after white light resection in all these patients. This was newly diagnosed and recurrent, and tumor tissue was sent to histopathology confirming residual tumor and tissue fluorescence. When comparing 5-ALA fluorescence-guided surgery and neuronavigation, an important study was published by Ponciani et al. in 2011. In this study, there were 23 glioblastoma patients, and sampling was performed both within and outside the contrast-enhancing neuronavigation boundary. In this study, non-fluorescent sampling within and outside the contrast-enhancing neuronavigation boundary was performed. In the results, you can see here the areas where there was tissue fluorescence. There were 41 specimens that had malignant glioma tissue present. The positive predictive value was 89 percent. This is the only study that I've found where the positive predictive value was in the 80s. Eighty-nine percentile. Most of the other studies are in the high 90s, but this study is important because, as you see here in the middle of the slide, tissue was sampled within the neuronavigation area, and the positive predictive value of those specimens was 100 percent. Now, when tissue was sampled outside the contrast-enhancing neuronavigation area, the positive predictive value fell, but that is because of the infiltrating biology of the tumor in those specimens that were sampled. False-positive fluorescence is a topic we will be covering now with 5-ALA fluorescence-guided surgery. False-positive fluorescence has been reported. It's really within the immediate vicinity of viable tumor cells. There have not been reports of false-positive fluorescence in normal brain distant from gross tumor. False-positive fluorescence has been described in patients with recurrent malignant gliomas who have completed prior adjuvant therapies. There may be reactive astrocytes at those areas that may metabolize 5-ALA to protoporphyrin 9. Autofluorescence has been described as well with any fluorophore. This may be a rare cause of false-positive fluorescence, and fluorescence can occur with radiation necrosis. We do know that with radiation necrosis and treatment effect, there still are tumor cells present within this. False-negative fluorescence has been also reported. There are several reasons and mechanisms that are felt to be a result of false-negative fluorescence. First, when there's malignant glioma tissue present and not visible by 5-ALA-induced fluorescence. This is likely due to the infiltrative biology of malignant gliomas. As we move away from the tumor, contrast-enhancing bulk, infiltrative biology of the tumor, results in less fluorescence away from the bulk and towards the margin. We do know that from the spectroscopy studies, the one I described and others that have been described, that we can identify weak fluorescence in these low-density cell infiltration zones that are too weak to visualize with a microscope. Confocal microscopy that's been reported by the BNI group, Dr. Natterson and I, can also be used to identify areas where there's weak fluorescence that cannot be visualized with a microscope, especially in low-grade gliomas. That's been published in 2007. Other reasons for false-negative fluorescence could be photobleaching, the presence of blood-covering tissue fluorescence and overhanging brain tissue. It's important to note that during fluorescence-guided surgery, to thoroughly examine the resection cavity and using the microscope to visualize under-overhanging brain tissue, suction blood to visualize the resection cavity properly, suction. The timing of 5-ALA administration may be another reason for false-negative fluorescence. Administering the 5-ALA agent and taking the patient to surgery too early may be the reason for false-negative fluorescence. However, taking the patient too late to surgery may not be as big of a problem as tumor fluorescence can be visualized up to 12 hours after surgery. Typically, with 5-ALA fluorescence-guided surgery, since a three-hour time window is usually required for proper tumor visualization, perform the surgery as the first case in the morning, patient arrives for their elective surgery on the same day. I would like to discuss the extent of resection studies that have been performed with 5-ALA fluorescence-guided surgery. This was a landmark, randomized Phase III study that was completed by Dr. Walter Stumer and multiple centers in Germany, published in Lancet Oncology in 2006. Other study results published in the Journal of Neurosurgery in 2010. In that randomized Phase III study, there was a significant increase in the extent of complete resection of patients who underwent 5-ALA administration. 63.6% of patients underwent a complete resection that was defined by their group as a residual tumor volume of less than 0.1 centimeters cubed of contrast in comparison to 37.6% of patients who underwent standard conventional white light surgery. Now, the other finding in this Phase III randomized study did show that progression-free survival at six months was also better in the 5-ALA fluorescence-guided surgery group in comparison to the conventional white light surgery group. Of note in the study, the majority of patients did not undergo the current standard of care with the stooped temozolomide chemoradiation regimen. And also, in this study, there were patients who did not undergo a neuronavigation section. There have been multiple studies looking at the extent of resection with 5-ALA fluorescence-guided surgery in high-grade gliomas. And mainly in these studies, the ability to resect the contrast-enhancing portion of the tumor is looked at. And you can see here that the complete resection of the contrast-enhancing tumor ranges from 80% to 89%. When looking at volumetric extent of resection, the extent of resection is in the 90s in many of these studies, 5-ALA is administered. We have recently reported in our initial group of patients that were enrolled in the Phase 2 study with 5-ALA, the ability to increase our extent of resection from 86% to 95%. We were able to look at our control patients in comparison to our 5-ALA patients. We've now treated close to 80 malignantly ill patients. In our study, which we recently published, we were able to look at the semi-automated volumetric and morphologic assessment prior and after glioblastoma resection with fluorescence guidance. This was able to allow for a more sensitive detection of resection using a semi-automated volumetric software that was developed. You can see here between October of 2011 and December of 2014, 55 patients were enrolled in the Phase 2 5-ALA fluorescence guided surgery trial at Emory University. We did have 4 patients excluded because of other pathologies. We examined 30 newly diagnosed glioblastoma patients. However, in our trial, we did have recurrent glioblastoma patients. We also had recurrent and newly diagnosed WHO grade 3 glioma patients. In our study, however, we only analyzed the newly diagnosed glioblastoma patients. They were the largest and most common cohort. We were very interested in understanding the resection and survival endpoints in these patients. Very sensitive extent of resection calculations were determined in our patients. We had very sensitive imaging, both pre- and post-operatively, in all our patients. We also assessed the morphological indices of the tumor prior to surgery, including the surface area of the tumor, percent of necrosis of the tumor volume, and the tumor surface area to volume ratio. We monitored patients by the standard cranial criteria. Both univariate and multivariate analyses were performed to evaluate the extent of resection and the residual tumor volume in our patients. Progression-free survival and overall survival were evaluated in a multivariate Cox regression model, and corresponding hazard ratios were reported with 25% confidence. In this slide, you can see here the preoperative tumor volumes that were assessed in a three-dimensional fashion by our semi-automated volumetric software. These were large tumors located in eloquent areas, and confirmed the abnormal surface area of the tumors that are not circumscribed as other tumor types. The median extent of resection in these 30 newly diagnosed glioblastoma patients was 94.3%. The median residual tumor volume was 0.821 centimeters cubed. These results were different than other results reported. We believe that the software we were able to use was very sensitive in detecting residual contrast enhancement. When defining complete resection as our European colleagues have defined with less than 0.175 centimeters cubed, we were able to achieve a complete resection in 30% of our patients. However, using the more general gross total resection concept of less than one centimeter cubed of contrast enhancement, we were able to achieve a gross total resection in 16, 53% of patients. We found that in our multivariate analysis, there was effect on overall survival with residual tumor volume, as well as the surface area volume ratio. Also, the MGMT promoter methylation status of the patient did have an impact on overall survival. Our phase two interim conclusions, by utilizing a very sophisticated semi-automated tumor segmentation technique, we found that in our prospective single arm clinical trial, that 5-ALA fluorescence guided surgery did permit more complete resection of our glioblastoma patient's tumor. We did find a greater overall survival in these patients with CBM. We also learned some new things that we are currently studying more, and that is the tumor shape and complexity can potentially outperform other methods for predicting the resectability of contrast enhancing portions of these tumors. We feel that these morphologic indices, such as tumor shape and composition, not only can impact resection, but could also impact patient survival. I'd like to switch gears now and discuss how 5-ALA can be used in diffuse infiltrating gliomas that are predominantly non-contrast enhancing. In this study that was published in 2010, patients who had non-significant contrast enhancing glioma, diffuse infiltrating gliomas, were administered 5-ALA. In those tumors, anaplastic foci were discovered with areas of tumor fluorescence that permitted the proper diagnosis of these patients as anaplastic gliomas. I think this is an important concept and topic. In tumors that we feel are lower grade WHO criteria due to their non-contrast enhancing state, please note that in these tumors there is a good chance that they do have anaplastic foci present. In a larger study published by the same group, this was studied in more detail, 59 patients, and really showed that anaplastic foci was present in a significant number of patients with non-significant contrast enhancement, altering their pathology from a grade 2 to a WHO grade 3 anaplastic astrocyte tumor. As you can imagine, this impacts the treatment of the patient who now would receive the STOOP regimen. This figure from that paper is showing you the non-contrast enhancing diffuse infiltrating in a lower figure G. This is a flare showing the diffuse tumor. In this series, imaging was performed using chemical shift as well as PET imaging to localize these areas and detect the anaplastic foci intraoperatively with visualization of protoporphyrin 9. The imaging allowed for localization of these areas, and this was performed histopathologically with multiple specimen trials. An important topic to cover as well is neurologic deficits that can arise from 5AA fluorescence guided surgery. We know that in the phase 3 randomized study, that at 6 weeks, there was really no significant difference between the 5-ALA group versus the conventional white light group. We do tend to see greater neurologic deficits immediately post-op, however, this balances out. Mark, over 50,000 patients have now been treated with 5-ALA. So the safety of 5-ALA, the safety profile of 5-ALA has been documented well, discussed with you some of the liver function test elevations that can occur in patients including the epithelial metabolism, skin sensitivity, those are really the main side effects of the drug in addition to nausea. I would like to conclude with some of the benefits of 5-ALA and move into the regulatory state of 5-ALA in the United States, but in conclusion for the benefits of 5-ALA fluorescence guided surgery in malignant gliomas, 5-ALA and protoporphyrin 9 intraoperative fluorescence is highly diagnostic of malignant tissue, really provides intraoperative visualization in real time to the neurosurgeon independent of brain shift and neuro-navigation, leads to more complete resection of malignant gliomas, possibly a greater increase in overall survival. It leads to identification of anaplastic foci and non-enhancing diffuse infiltrated gliomas and we know this is a safe agent for our patients. Now I'd like to move in just to a brief overview of the regulatory state of 5-ALA in the United States. It can only be used with an IND through the FDA currently, it is not approved for use in the United States. I do want to let you know however the FDA has been approached multiple times and we are on a path towards a new drug application for FDA approval. We did approach the FDA in 2011 and presented the European clinical experience with safety and progression-free survival increase, at that time they did want us to show clinical benefit in our patients, we have now met with the FDA again and just to bring up to the group there are training sessions that have been available and will be available for neurosurgeons to use 5-ALA in the United States and this will be a requirement prior to any use for obtaining drug in the United States. 5-ALA has obtained FDA orphan drug approval in 2013, the Gliolan formulation which is approved in Europe and Asia is what obtained orphan drug approval. The FDA was approached again in September 2014 and they have given the go-ahead for an FDA new drug application. There was also a pre-NDA meeting with the FDA in June of 2014. The proposed indication for Gliolan in the United States will be Gliolan is an imaging agent to facilitate the real-time detection and visualization of malignant tissue during Gliolan surgery. The submission of the FDA NDA is currently going on, there is a large movement to make this happen, as you can imagine this requires a lot of effort from multiple parties, NX Corporation in collaboration with others is leading this effort in the United States. We have released a multi-center trial to really assess tissue fluorescence and malignancy and assess the positive predictive value in a multi-center trial here in the United States. Please reach out to me if you have any questions of this trial, we've communicated to many already on this trial and we hope to launch as soon as possible. I would like to acknowledge NX Development, Dr. Alan Esbren, Photonamik who provides the drug from Germany, Dr. Anne Moore, Ulrich Kosiesa, Brad Karadov for showing some of the data initially with the in vitro studies, and Professor Dr. Shuming Ni. I do want to acknowledge Dr. Scott Cordova, MD PhD candidate at Emory who did a lot of the volumetric work I presented, Dr. Hyun-Suk Shim, and then members of my laboratory. Thank you very much. At this point, I leave you my contact information. I would like to now go over the questions. I want to make sure that we answer these questions completely. Please submit your questions and we will go over them one by one. I have a question here, how can one be sure that none of the tumor cells were left behind? Can't there be some glial cells which do not fluoresce? This goes back to the points we made about the positive predictive value of 5-ALA induced tissue fluorescence in tumor presence, which is 90s, high 90s. There have been reports of false tissue fluorescence at the margin that could be reactive astrocytes, but this is within the immediate vicinity of the tumor. We can confidently resect the violet, red, and pink tissue knowing that that represents tumors. We have a question here, how does the vascular control, I'm not quite sure what that question means. The 5-ALA does rely on the blood-brain barrier breakdown, however, it does cross the blood-brain barrier into the area outside of contrast enhancement present on MRI scans. The flip side of this, is this tumor, is this the corticospinal tract? Recent publications have suggested integrating electrical stimulation with either regular or acoustic suction to detect proximity to the corticospinal tract in real time during tumor resection. How would you handle a situation in which 5-ALA showed tumor fluorescence at the same location where a low electrical threshold indicated close proximity to motor tract? This is a great question. I think we know that a small percentage of patients, there are corticospinal tracts that run through the tumor that has been published by other studies. I think that using correct neurosurgical judgment and our tools available to us, 5-ALA and fluorescence guided surgery is a tool, just as corticospinal tract monitoring with correct stimulation is a tool as well. I think combining these tools is very effective and very helpful in really resecting what is tumor and leaving behind eloquent motor tracts. I hope I answered that question correctly. It has been reported subappendal fluorescence in tumor-free areas. Is there a particular reason? There has been subappendal fluorescence detected in malignant glial tumors that has spread along the subappendal area. However, there has been some reports of subappendal fluorescence in the tumor. Will recurrent GBMs affect the diagnostic accuracy of fluorescence guided surgery? No, it does not. In the NABABI study we went over, the positive predictive value is still very high with recurrent glioblastomas. Of course, the fear is a tissue that has been treated with chemoradiation, could that permit for nonspecific uptake of 5-ALA? The studies that have come out so far with recurrent glioblastoma really support the use of 5-ALA in recurrent gliomas. Is there any study that supports an increase in the survival of fluorescence guided surgery against only navigation? I am not aware of any study on that. I will have to look that up, see if we can answer that. I am not aware of a study that looks at survival of fluorescence guided surgery navigation. We have Dr. Cabrera Aldana from Mexico. We would like to know if the lesions of all the 30 high grade glioma patients from your study were amenable to gross total resection or included all kinds of location? Excellent question. In the paper, if you are able to pull it up and I can send it to you, the majority of our patients had eloquent located tumors. Over 80% of our patients' tumors were all eloquent location. They were amenable, many of them, to gross total resection. However, many of them were not amenable to gross total resection. That also contributed to our lower complete resection rate. Good question. What are the risks, complications, or contraindications for glioland? Anyone who has a history of porphyria, for obvious reasons, would be ineligible for the operation of glioland. Anyone, obviously, who is pregnant cannot get glioland. We don't know the risk of fetus with glioland. I think we discussed some of the safety issues. There are a significant number of patients who do have liver function test abnormalities. All of them go back to normal within two months after the oral administration of 5-ALA. The neurologic deficits we discussed, they usually balance out after four to six weeks after surgery. I think those are some of the main things that we discussed with 5-ALA. How do I procure 5-ALA in the USA today? I think I discussed with you that we do have a multicenter study under my IND that is currently being released. Please reach out to me, and I will contact with the Annex Development Corporation, who is helping with the study. It is an investigator-initiated study under my IND. Any other tumors besides gliomas react to 5-ALA? There are case reports of lymphoma, yes. There are reports of sub-ependymomas, reports of ependymomas, intramedullary location of the spinal cord. Even demyelinating plaques can actually fluoresce with 5-ALA. Those are some of the tumors. Meningiomas, the majority of meningiomas will fluoresce in addition to the hyperostatic pulmonary involvement, documented in multiple publications. Also, metastatic tumors, approximately 50 to 60% of metastatic tumors will fluoresce after 5-ALA administration. What is the real cost of 5-ALA for surgery? Great question. In Europe, there is no cost in our clinical trial here. It is provided free by the company in Germany that produces it, Photonomic. In Europe, I know that they charge between 1,000 and 2,000 euros for treatment per vial of CLN. Describe the time process it takes to capture a single 5-ALA video and play back. How does it impact on the flow of surgery? When we switch over on the microscope, it takes about a half a second with the push of a button or the lever to activate the blue light. You will be able to visualize the tissue fluorescence in real-time through the binoculars of the microscope. We are not visualizing this on the monitor. This is not near-infrared visualization such as ICG, where you have to look at a monitor. You are looking through the binoculars of the microscope in real-time with blue light at the tissue. How cumbersome is the 3-5 hour administration of 5-ALA? An 8 a.m. operation needs 5 a.m. administration. That is a challenge. If you can bring your patient in early to administer the agent, that is great. Most commonly, the drug is administered when the pharmacist arrives, who has to mix the formulation, which typically is 6.30 to 7.00. We are talking about a case that starts at 10.30. The formulation can be mixed the night prior to surgery. If the surgery is delayed, we do not dose the glialand again. The beauty of glialand in comparison to other fluorescent agents is that it does hang out in the tumor tissue for up to 12 hours. Typically, you will not be delayed 12 hours, but you could be delayed 5 hours or so during surgery. No worries. You will still be able to visualize the tumor. I have done that in a number of cases where I was delayed by an emergency and the tumor fluoresced 5-6 hours after the operation. If we have taken the glialand course and have a blue light source for our microscope and received IRB approval, when can we actually use glialand? I think you will be able to use glialand very soon thereafter. Please maintain contact with the company on this. The only rate limiting step right now is since I have moved to Mount Sinai, the protocol is being evaluated by the IRB here. Once we are IRB approved at Mount Sinai, we will be able to initiate the studies in those centers that are IRB approved with the microscope platform. Is there any naturally producing protoporphyrin 9 in the glioma or brain tissue? Again, protoporphyrin 9 is present in our body right now. The answer to the question is yes. What we do with the administration of 5A-LA is we are overloading the system. Because we believe that the ferroquilatase enzyme is less in malignant gliomas, the protoporphyrin 9 accumulates more. When the FDA approves other medications for use, they do not seem to require training to use the newly approved drug. Why is glialand different? That is a very good question. I think it is a very important answer. I think there are a lot of nuances with glialand and 5A-LA fluorescence guided surgery that require an extra training. I think it is important to understand the visualization of the fluorescence guided surgery, understand its limitations, understand the safety profile, which we did go over in this webinar, but there are important presentations that need to be viewed to go into this in more detail prior to going and treating patients. Remember, this is an oral agent that is given prior to surgery. It requires a specialized microscope that has been fitted with a fluorescent filter. There are some other important techniques that need to be understood prior to going to the patient. How can fluorescence guided surgery help with the complete resection of the tumor in areas such as the motor area, especially owing to the fact that the tumor resection will also mean that some non-tumor area will also need to be removed? I think this goes with an earlier question that was proposed. Using intraoperative mapping techniques with 5A-LA I think is a very powerful set of tools that will help the neurosurgeon identify these elephant pathways and then also visualize in real time the fluorescence clinically on the tissue. There is no reason to take non-tumor area. The fluorescence guided tissue really denotes tumor. However, as we move out past the tumor margin, we are going into infiltrating tumor where there are normal cells. As we push our resection out past or including the pink vague fluorescent region, then there could be some impact on these motor tracts. That's where the cortical mapping can help us steer away from those areas that are eloquent. Was the worsened neurologic deficit early after surgery due to more aggressive resection? I think the answer to that question is yes. I think that's the universal question with the Phase 3 study. In our experience with our Phase 2 study, we didn't see that as often as it occurred in the Phase 3 study. I think in the Phase 3 randomized study that was the answer. Are there any skin precautions post-op? Yes. We want to keep the patient for 24 to 48 hours. We usually go up to 48 hours. We keep the patient in a dimly light area. Certainly, there's been reports of in the operating room, shining the surgical light on the skin could cause skin sensitivity. It results in a skin sunburn-like effect. With the patients there, the lights are kept dim. Best case scenario, how long before this will be available in the U.S. for use without IRB? That's a tough question. The new drug application is going in very soon for expedited review. It could be within 12 months. Requirement for training, but something like Gliolin is much more dangerous and toxic to both patient and OR staff. No special training is required. Again, good point. I don't want to compare Gliolin and 5-ALA to Gliadel. I think that's a chemotherapeutic agent. 5-ALA is not a therapeutic agent. It's an intraoperative imaging agent. It is orally consumed, and it is used in combination with surgery. The training, I think it's a very important thing with any technology to be properly trained. I don't think we can avoid the training. Last but not least, 5-ALA has the same effectiveness as pediatric gliomas and or posterior fossa gliomas. This area has a lot of future research to be done, but we think that in the pediatric gliomas there's going to be a huge impact because we know the extent of resection of pediatric gliomas is associated with longer overall survival. I think this is going to be an important topic that we will see studied. Currently, there's not a lot of safety data for pediatric patients, so that's slowly being accumulated. There has been some pediatric patients treated in the United States with another Glioland formulation early on, and certainly in Europe there's pediatric patients undergoing surgery. How much time is necessary to do the training? The training is really a one-day training, less than one day. It starts in the morning, it finishes around noon, and then a trainee comes to the operating room to see a case in real time. What is the cost of the filter? Is it available for Zeiss microscopes? Yes, it's available for Leica and for Zeiss. You can have your current microscope modified. You can talk to your local Zeiss or Leica rep, and they will discuss that with you. You can also purchase the microscope with them. Is there potential to develop an intravenous form of 5-ALA to eliminate the pre-op administration strain? There really is no need for an IV form. It's absorbed so rapidly within the gut that there is no reason to develop an IV form. Yes, is there a particular reason for autofluorescence in the ependymal wall? I think there are case reports of this, and I don't think I have a good answer for that, but unfortunately it's a limited number of cases. Okay, let's see here. We have one question that just popped up here. Have you ever tried to correlate 5-ALA intensity fluorescence with the phenotype of GBM? It could be a really interesting issue. Yes, I agree. We are doing that. We're actually quantifying fluorescence intensity and then looking at the specific histopathology of the tumor. We're also looking within the tumor, each different environment of the tumor, the necrotic core. We're looking at the tumor bulk. We're looking at the margin and trying to understand these different areas with regards to tissue fluorescence and even genomics and proteomics. Okay, well I think we answered all the questions. Thank you once again for taking the time to participate in the webinar tonight. I do want to again thank Leica for sponsoring this webinar, and I especially want to thank the AANS and the NREF for the webinar tonight. Please, the NREF is an important part of the AANS that permits high-caliber, high-quality research to be done by our residents. Please consider this with future.

5-ALA fluorescence-guided surgery

brain tumors

Dr. Haja Panias

malignant brain tissue

extent of resection

safety of 5-ALA

advantages

real-time visualization

malignant gliomas

limitations

healthcare professionals