Intraoperative 5-ALA fluorescence-guided resection of high-grade glioma leads to greater extent of resection with better outcomes: a systematic review

High-grade gliomas (HGG) are the most aggressive and common malignant brain tumors in adults. They have a dismally fatal prognosis. Even if gross total resection of the enhancing tumor is achieved, inevitably, invading tumor cells that are indistinguishable to the un-aided eye are left behind, which eventually leads to tumor recurrence. 5-aminolevulinic acid (5-ALA) is an increasingly utilized intraoperative fluorescent imaging agent for patients with HGG. It enhances visualization of HGG tissue. Despite early promising randomized clinical trial data suggesting a survival benefit for 5-ALA-guided surgery, the growing body of literature must be analyzed to confirm efficacy on patient outcomes. To perform a systematic review of the literature to evaluate whether there is a beneficial effect upon survival and extent of resection due to the utilization of 5-ALA in HGG surgery. Literature regarding 5-ALA usage in HGG surgery was reviewed according to the PRISMA guidelines. Two databases, PubMed and SCOPUS, were searched for assorted combinations of the keywords “5-ALA,” “high-grade glioma,” “5-aminolevulinic acid,” and “resection” in July 2020 for case reports and retrospective, prospective, and randomized clinical trials assessing and analyzing 5-ALA intraoperative use in patients with HGG. Entailed studies on PubMed and SCOPUS were found for screening using a snowball search technique upon the initially searched papers. Systematic reviews and meta-analyses were excluded from our PRISMA table. 3756 previously published studies were screened, 536 of which were further evaluated, and ultimately 45 were included in our systematic review. There were no date restrictions on the screened publications. Our literature search was finalized on July 16, 2020. We found an observed increase in the overall survival (OS) and progression-free survival (PFS) of the 5-ALA group compared to the white light group, as well as an observed increase in the OS and PFS of complete resections compared to incomplete resections. Of the studies that directly compared the use of 5-ALA to white light (13 of the total analyzed 45, or 28.9%), 5-ALA lead to a better PFS and OS in 88.4 and 67.5% of patients, respectively. When the studies that reported postoperative neurologic outcomes of surgeries using 5-ALA vs. white light were analyzed, 42.2% of subjects demonstrated 5-ALA use was associated with less post-op neurological deficits, whereas 34.5% demonstrated no difference between 5-ALA and without. 23.3% of studies showed that intraoperative 5-ALA guided surgeries lead to more post-op neurological deficits. Utilization of 5-ALA was found to be associated with a greater extent of resection in HGG surgeries, as well as longer OS and PFS. Postop neurologic deficit rates were mixed and inconclusive when comparing 5-ALA groups to white light groups. 5-ALA is a useful surgical adjunct for resection of HGG when patient safety is preserved.


Introduction
High grade gliomas (HGG) are tumors of the glial cells in the central nervous system. These gliomas are referred to as high grade because they are rapidly growing and categorized as WHO grade III or IV tumors. HGG are the most aggressive and common types of brain tumors in adults. They include gliosarcomas, anaplastic astrocytomas, oligoastrocytomas, diffuse brainstem gliomas/diffuse pontine gliomas, pleiomorphic xanthoastrocytomas, and glioblastoma multiforme (GBM). Despite surgical resection, radiation therapy, and chemotherapy, microscopic residual tumor is inevitable due to the highly invasive nature of HGG, which leads to tumor recurrence.
One emerging tool in the neurosurgical arsenal is using intraoperative 5-aminolevulinic acid (5-ALA) for fluorescence-guided surgery to maximize tumor resection and minimize residual tumor. In 2017, the U.S. Food and Drug Administration (FDA) approved 5-ALA for use as an intraoperative fluorescent imaging agent in patients with HGG [1]. 5-ALA enhances visualization of malignant brain tumor tissue to potentially result in a more complete resection of the tumor. Approximately a decade prior to its approval in the United States, it was first approved in Europe by the European Medicine's Agency (EMA) in 2007 [1]. The European approval followed Dr. Walter Stummer's 2006 randomized controlled multicenter phase III trial which substantiated that 5-ALA can lead to more complete HGG resection with better outcomes and greater 6 month progression free survival than white light visualization alone [2]. Stummer's work was a landmark paper describing a survival benefit from the use of 5-ALA. High-grade glioma patients fare better in terms of survival with gross-total resection [3]. The greater the extent of resection, the longer the overall survival of the patient [2]. Intraoperative 5-ALA can potentially help maximize tumor resection.
Currently, this solution is inadequately investigated, as it has been 14 years since Stummer's paper was written, and there has not been much generalized conclusion regarding the survival benefit from the use of 5-ALA in GBM resection. However, during the past 14 years, a copious amount of papers have been written about individual 5-ALA experiences. It is imperative to analyze these papers to see if they confirm the benefit observed in the Stummer 2006 study. Additionally, it appears that although 5-ALA is associated with a greater extent of resection, its use may be associated with a potential increase in post-op complication/neurological deficit. This postulation must be further explored as well.

Search strategy
Two databases, PubMed and SCOPUS, were searched in July 2020 for case reports, retrospective, prospective, and randomized clinical trials assessing and analyzing 5-ALA intraoperative use in patients with HGG. Entailed studies on PubMed and SCOPUS were found for screening using a snowball search technique upon the initially searched papers. Systematic reviews and meta-analyses were excluded from our PRISMA table. Assorted combinations of the keywords "5-ALA," "high-grade glioma," "5-aminolevulinic acid," and "resection" were used in the search. Our systematic review was reported in accordance to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. [4] We performed searches in PubMed and SCO-PUS from inception to July 16, 2020. Case reports, retrospective, prospective, and randomized clinical trials evaluating the application, characteristics, and effects of 5-ALA use in HGG resection were included. There was no limit by year of publication. Editorials were excluded. The selection of sources was agreed upon by consensus. The first author, year, study design, country, number of participants, figures, content, diagnosis, pathology, prognosis, and timeline were all included data taken into consideration.

Study selection
The included studies had to definitively use 5-ALA as an intraoperative imaging agent upon HGG neurosurgery. 3756 full-text studies were assessed for eligibility and inclusion in this systematic review (Fig. 1). 2029 studies were initially excluded due to complete irrelevance and duplication. Upon screening the remaining 1727 abstracts, a further 1191 studies were excluded for substantial irrelevance. Of the 536 remaining full texts that were read, 491 were excluded for the following reasons: limited relevance, lowgrade gliomas (WHO grade I and II), focus upon 5-ALA sensitivity, specificity, PPV, and NPV, and failure to include statistics/results regarding survival, extent of resection, and percent gross-total resection. Thus, 45 studies were included (Table 1).

Study characteristics
The reviewed studies included 21 prospective, 20 retrospective, 3 randomized phase trials, and 1 parallel, randomized, balanced, group-sequential, two-armed, controlled multicenter phase III study. A total of 3756 titles and abstracts were identified by the aforementioned search tactics, of which 45 met our inclusion criteria and were included in the qualitative synthesis. The total number of patients across all of the 45 included studies were at least 4599 (Table 1). One study by Stepp et al. [5] was carried out on patients at 18 clinics in Germany, however, the specific amount of patients was not specified in the text. The 45 studies all used 5-ALA to remove HGG tumors and often focused on 5-ALA vs. white light, 5-ALA only, and 5-ALA combined with technologies, such as intraoperative MRI (iMRI), contrast-enhanced ultrasound (CEUS), intraoperative ultrasound (ioUS), and intraoperative CT (i-CT).
Of the 45 analyzed studies, 13 of these studies (28.9%) compared HGG resection guided by 5-ALA vs. white light. These 13 studies totaled 1984 patients. Of the 1984 patients, a total of 1525 patients were analyzed for progression-free survival (PFS), 1616 analyzed for overall survival (OS), and 1077 explored post-op neurological deficit. Regarding PFS,
The combination of 5-ALA with different intraoperative technological modalities yielded mixed results. Barbagallo et al. [39] did not find a statistically significant difference between the PFS and OS of intraoperative 5-ALA usage only versus 5-ALA + i-CT. A study by Coburger et al. [40] demonstrated that the combination of iMRI + 5-ALA versus iMRI without 5-ALA led to a greater amount of GTR and a greater OS. Similar studies by Eyüpoglu et al. [41] and Nickel et al. [42] both demonstrate that iMRI + 5-ALA led to a higher EOR as opposed to 5-ALA alone. Conversely, Tsugu et al. [43] concluded that 5-ALA alone resulted in a higher percentage of complete resection, but a lower EOR than iMRI + 5-ALA. Roder et al. [44] showed that surgery solely with iMRI led to a higher percentage of GTR, greater EOR, and a higher PFS than surgery solely with 5-ALA. Surgery guided by iMRI versus non-iMRI in a study by Schatlo et al. [45] indicated that iMRI usage led to a higher overall survival. Yamada et al. [46] reported an impressive amount of 95% complete resections executed by 5-ALA + iMRI. Additionally, Della Pepa et al. [47] studied the combined use of intraoperative 5-ALA + CEUS and found that the combination resulted in the highest EOR as compared with 5-ALA alone, CEUS alone, and conventional surgical technique. Neidert et al. [48] found that 5-ALA used in conjunction with ioUS resulted in a higher OS than 5-ALA alone.
Overall, OS and PFS were longer in the 5-ALA groups when compared to white light groups in studies individually, as well as complete resection groups to incomplete resection groups. The most common post-operative neurological deficits were motor, language, and visual in nature. They consisted more specifically of aphasia, hemiparesis, and hemianopsia. Post-op seizures and hemorrhages were also observed.
There could be an assumption that higher neurological complication rate of HGG resection using 5-ALA as compared to white light would occur due to its more aggressive surgical resection. Yet, the included studies were rather inconclusive and disparate regarding if 5-ALA led to more complications. Across the studies that included complication rates, 5-ALA was better that white light in 454 patients (42.2%), worse in 251 (23.3%), and showed no statistical difference between 5-ALA and white light in 372 patients (34.5%). In multiple studies, there were surgeries that were halted, leaving behind residual 5-ALAfluorescing tissue in order to avoid causing neurological deficit. For example, in the study by Chan et al., minimal residual 5-ALA fluorescence was left in three patients to avoid post-operative deficit [31]. Della Puppa et al. stopped surgery in 26% of cases to avoid neurological deficits [22]; Feigl et al. stopped 24% of surgeries, leaving residual 5-ALA fluorescence to avoid deficits [35]. Jacquesson et al. noted leaving 5-ALA fluorescence to avoid post-operative neurological deficits as well in 31.8% of their cases [16].
0% of cases reported an allergy to 5-ALA reported. If a patient is known to have an allergy to 5-ALA or is an extremely atopic individual, proper prophylaxis is necessary to avoid allergenic complications. Or rather, as seen in studies such as Chan et al., 5-ALA was contraindicated in patients with an allergy to it [31]. Allergies to 5-ALA are rare, and anaphylaxis is incredibly rare. Supposedly, the first, and seemingly only, case of severe allergic reaction (anaphylaxis) to intravesical instillation of hexaminolevulinate hydrochloride in literature was reported by Colapaoli et al. [49]. However, 5-ALA in this case was not used for HGG resection, but rather for fluorescence cystoscopy. Generally, neurosurgeons utilizing 5-ALA to guide HGG resections should be very conscientious of fluorescing functional, healthy tumor. It is important to note the need to respect the boundaries of the actual HGG tissue and proceed with proper caution.

Discussion
Our analysis has demonstrated that 5-ALA is beneficial compared to white light alone. Additionally, our review has shown that gross total resection compared to incomplete resection favors longer PFS and OS across individual studies. These results are consistent with published findings in the literature showing that gross total, but not incomplete, resection of GBM prolongs survival in conjunction with radiochemotherapy [50]. Overall, OS and PFS of the 5-ALA group tended to be longer compared to the white light group, as well as longer after complete resection compared to incomplete resection. Of the studies that directly compared the use of 5-ALA to the lack thereof (13 of the total analyzed 45, or 28.9%), 5-ALA led to a better PFS and OS in 88.4% and 67.5% of patients, respectively ( Table 2).
The results from analyzing the included literature support the utilization of intraoperative 5-ALA FGR in the removal  [3,51]. Illuminating cancerous tissue and its borders so that the surgeon can visualize what he/she must remove would sensibly lead to a more effective resection.
Combining intraoperative technological modalities with intraoperative 5-ALA utilization correlates with higher survival than does 5-ALA, iMRI, i-CT, ioUS, or CEUS each alone. This finding substantiates the need to further investigate the combination of fluorescent and technical modalities to potentially reach maximal survival rates and EOR. Such a combination may be the most optimized modality for HGG removal known to date.
There are some potential drawbacks to the use of intraoperative 5-ALA that should be considered. It appears that 5-ALA can help achieve a more complete resection, but with this comes a slight increase in post-op complication/ neurological deficit. The reasoning behind this may be that the greater the amount resected, the higher the risk of excising healthy, functional tissue. There exists research positing that 5-ALA may enable extension of tumor resection beyond radiologically evident tumor. This extension may potentially put intact, functional adjacent tissue at risk of destruction. Therefore, 5-ALA may lead surgeons to resect too far beyond the HGG tumor's borders and damage healthy, eloquent brain matter [52]. This risk may explain the discrepancy in numbers of post-op neurological deficit reported. It is important to note that due to the increased vigilance and more conservative resection executed in these studies, we believe that the neurological complication rate due to the use of 5-ALA in HGG resection was far lower than it would have been if the neurosurgeons had not preventively acted with the responsible foresight with which they did. Multiple studies documented the halting of surgeries as to not aggressively resect tissue beyond the borders of the HGG.
The mixed findings regarding the effect of 5-ALA use on post-operative neurological deficit complicate interpretation of these studies. It is possible that differences in tumor location, surgeon skill, and patient medical comorbidities may confound neurologic deficit data. Thus, no clear conclusion regarding the risk of 5-ALA florescence guided surgery on neurologic deficit can be reached. We recommend that surgeons use vigilance when operating near eloquent brain areas, particularly when relying on 5-ALA fluorescence-guided surgery, to optimize patient safety. However, more data is needed to properly decipher whether or not 5-ALA-guided HGG resection causes a statistically significant increase in neurological deficits. The extent of deficit is partially reliant on the skill of surgeon and the eloquence of the areas in which the tumor sits. Postoperative deficits could both be caused by surgeons resecting beyond the boundaries of fluorescent tumor and by 5-ALA highlighting functional brain tissue. Neurological deficits when using 5-ALA can be minimized by using imaging techniques, such as intraoperative neurophysiological monitoring, ultrasound, MRI, and iCT.
Additionally, 5-ALA used intraoperatively has been rarely found to be below fluorescing threshold. In this instance, 5-ALA succeeds in tissue uptake but fails in properly and effectively fluorescing. This failure could inhibit the surgeon from seeing the borders of the tumor, eliminating the intended benefits of using intraoperative 5-ALA in the first place. Thus, intimate knowledge of neuroanatomy and frequent anatomic re-orientation must be utilized in conjunction with 5-ALA to avoid new neurologic deficits by injuring eloquent brain or white matter tracts.

Limitations
Limitations of our study exist. The included studies are associated with different sample sizes and methods of analyses. Due to the overall small sample sizes because of the novelty of 5-ALA intraoperative usage in HGG resection, especially in the United States, the ability to draw similarities between cases is somewhat limited. There is a high degree of heterogeneity between studies with respect to their quantitative outcomes and as such, pooling and quantitative analysis of the meta-data is inappropriate statistically. Until more studies are available that can help overcome the risk of small study and publication biases, meta-analysis of the data should be deferred for now and only the systematic trends between studies highlighted. Finally, the studies tend to be performed by high volume cranial surgeons at expert centers. Generalizability of these results to surgeons who perform few cranial surgeries each year must be exercised with caution.

Conclusion
Despite potential drawbacks, overall, utilization of 5-aminolevulinic acid (5-ALA) may ultimately lead to better outcomes and longer survival rates for patients who undergo 5-ALA FGR. An implication of this finding is that 5-ALA FGR may be a better alternative to conventional surgical resection of HGG. Combining 5-ALA with an additional technical modality, such as iMRI, i-CT, ioUS, or CEUS may be even more beneficial than 5-ALA alone. This possibility warrants further research.
The results regarding postoperative neurologic deficits of surgeries using 5-ALA compared to those using white light do not coincide enough with one another to arrive at a clear conclusion. The reasoning behind this may be that the greater the amount resected, the higher the risk of excising healthy, functional tissue. The extent of deficit appears to be partially reliant on the skill of surgeon and the eloquence of the areas in which the tumor sits. When using 5-ALA, neurological deficits can be minimized by using imaging techniques, such as intraoperative neurophysiological monitoring, ultrasound, MRI, and iCT.
Author contributions All authors contributed to the study conception and design. Material, preparation, data collection, and analysis were performed by TE, DE, and VML. The first draft of the manuscript was written by TE; DGE and MEI commented/edited and VML executed statistical/data analysis. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding This systematic review received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.

Data availability
The data that support the findings of this study are openly available at https:// pubmed. ncbi. nlm. nih. gov and https:// www. scopus. com/ home. uri.

Conflicts of interest
The following authors have no financial disclosures or personal conflicts of interest: TAE, DE, VL, LD, RJK, MEI.
Ethical approval Ethical approval was not applicable for this study as only publicly accessible data was utilized.