Neurosurgery Service, Alicante University General Hospital, Alicante Institute for Health and Biomedical Research (ISABIAL - FISABIO Foundation), Alicante, Spain.
5-ALA-based fluorescence guided surgery has been shown to be a safe and effective method to improve intraoperative visualization and resection of malignant gliomas. However, it remains ineffective in guiding the resection of lower-grade, non-enhancing, and deep-seated tumors, mainly because these tumors do not produce detectable fluorescence with conventional visualization technologies, namely, wide-field (WF) surgical microscope. The introduction of fluorescence guided resection (FGS) represents one of the most important advances in the neurosurgical treatment of brain tumors.
5 aminolevulinic acid fluorescence guided resection permits the intraoperative visualization of malignant glioma tissue and supports the neurosurgeon with real-time guidance for differentiating tumor from normal brain that is independent of neuronavigation and brain shift.
Wei et al., describe some of the main factors that limit the sensitivity and accuracy of conventional WF surgical microscopy, and then provide a survey of commercial and research prototypes being developed to address these challenges, along with their principles, advantages and disadvantages, as well as the current status of clinical translation for each technology. They also provide a neurosurgical perspective on how these visualization technologies might best be implemented for guiding glioma surgeries in the future
Detection of PpIX expression in low-grade gliomas and at the infiltrative margins of all gliomas has been achieved with high-sensitivity probe-based visualization techniques. Deep-tissue PpIX imaging of up to 5 mm has also been achieved using red-light illumination techniques. Spectroscopic approaches have enabled more accurate quantification of PpIX expression.
Advancements in visualization technologies have extended the sensitivity and accuracy of conventional WF surgical microscopy. These technologies will continue to be refined to further improve the extent of resection in glioma patients using 5-ALA-induced fluorescence 1).
Schebesch et al., from the University Medical Center Regensburg, Germany published five patients (3 female, 2 male; mean age 45.4 years) who underwent fluorescence-guided surgery for supratentorial, intracerebral lesions which showed no contrast-enhancement in the preoperative MRI but were, however, strongly suspicious for gliomas. Accordingly, all patients received a preoperative FET-PET scan and detailed histopathological workup was performed. After giving written informed consent, all patients received 5 mg/kg of FL at the induction of anesthesia. Surgery was conducted under white light and under the YELLOW 560 nm filter. We reviewed the surgical protocols, navigational storage and the image databases of our surgical microscopes for evidence of intraoperative fluorescence that corresponded to the FET-PET positive area.
In all patients they found distinct accordances between the FET-PET positive areas and the fluorescing regions within the targeted lesions. Histopathological workup of the fluorescent tissue revealed anaplastic oligodendroglioma, IDH-mutant and 1p/19-codeleted (WHO grade III) (n = 2), anaplastic astrocytoma, IDH-mutant (WHO grade III) (n = 1), oligodendroglioma, IDH-mutant and 1p/19q-codeleted (WHO grade II) (n = 1) and pilocytic astrocytoma (WHO grade I) (n = 1). No adverse events were noted.
Despite the lack of gadolinium-enhancement in the preoperative MRI, all patients intravenously received FL to guide resection. Irrespective of the final grading, FL was extremely helpful in detecting the lesions and in identifying their border zones. In selected patients with NEG, but strong metabolic activity according to the FET-PET, FL may significantly increase the accuracy of surgery 4).
5 aminolevulinic acid has initiated a radical change. Over the past years, various groups have published rates of complete resection of the enhancing tumor that exceed 80%. In the coming years, as the use of the technology expands, complete resection should become a common, predictable result at many centers. Consequently, adjuvant therapies that benefit from resection could play a bigger role, resection could be incorporated as a variable in randomized trials and distant recurrence might become a more common problem 5).
Fluorescence guided resection (FGR) in high grade glioma surgery aims at increasing complete resections and, thus, local control. This technique uses 5 aminolevulinic acid (5-ALA), a natural intermediate substance in the heme-porphyrin biosynthesis pathway, and a protoporphyrin IX (PpIX) precursor. PpIX is fluorescent under blue light exposure 6).
The highest visible and measurable fluorescence was yielded by 20 mg/kg. No fluorescence was elicited at 0.2 mg/kg. Increasing 5-ALA doses did not result in proportional increases in tissue fluorescence or PPIX accumulation in plasma, indicating that doses higher than 20 mg/kg will not elicit useful increases in fluorescence 7).
In 2000 Stummer et al published that Five-aminolevulinic acid (5-ALA)-derived fluorescence was approved for fluorescence-guided resections of malignant gliomas, relying on selective synthesis and accumulation of protoporphyrin IX (PPIX) within malignant glioma cells 8).
In 2006 Stummer et al., published that tumour fluorescence derived from 5 aminolevulinic acid enables more complete resections of contrast-enhancing tumour, leading to improved progression-free survival in patients with malignant glioma 9).
The positive predictive value (PPV) of utilizing the most robust ALA fluorescence intensity (lava-like orange) as a predictor of tumor presence is high. However, the negative predictive value (NPV) of utilizing the absence of fluorescence as an indicator of no tumor is poor. ALA intensity is a strong predictor for degree of tumor cellularity for the most fluorescent areas but less so for lower ALA intensities. Even in the absence of tumor cells, reactive changes may lead to ALA fluorescence 10).
Many studies have shown that the ratio of gross total resections was higher if the fluorescence technique was used. The fluorescence signal intensity is correlated to the cell density and the PpIX concentration. The current method has a very high specificity but still lower sensitivity, particularly regarding the zones with poor tumoral infiltration 11).
PpIX fluorescence was also identified as a novel marker for intraoperative detection of anaplastic foci in nonenhancing gliomas that ensures a precise histopathological diagnosis and optimal patient treatment. Furthermore, 5 prospective studies have confirmed that PpIX fluorescence is able to identify residual tumor tissue after assumed maximal resection of malignant gliomas with conventional white-light microscopy. Because intraoperative detection of malignant glioma tissue is significantly improved by 5-ALA FGS, high rates of complete tumor resections are achieved, especially in combination with intraoperative monitoring and mapping 12).
In the future, chemotherapy with new anticancer agents, immunotherapy, and new methods of radiotherapy and gene therapy will be developed; however, ALA will play a key role in malignant glioma treatment before the development of these new treatments 13).
Visual assessment of PpIX fluorescence is subjective and limited by the distorting effects of light attenuation and tissue autofluorescence 14).
Resection of reactive tissue without active recurrent tumor after multimodal treatment for glioblastoma is frequently associated with solid or vague 5-AIF. Therefore, neurosurgeons should remain cautious when attempting to employ intraoperative 5-ALA induced fluorescence (5-AIF) to discriminate radiation- and chemotherapy-induced tissue changes from true disease progression. Nevertheless, 5-AIF-guided resection remains a valid tool in the neurosurgical treatment of recurrent gliomas 15).
The resection cavity underestimates the volume of resected tissue and 5-ALA complete resections go significantly beyond the volume of pre-operative contrast-enhancing tumor bulk on MRI, indicating that 5-ALA also stains MRI non-enhancing tumor tissue. Use of 5-ALA may thus enable extension of coalescent tumor resection beyond radiologically evident tumor. The impact of this more extended resection method on time to progression and overall survival has not been determined, and potentially puts adjacent and functionally intact tissue at risk 16).
In 2013 based on available literature, there was level of evidence 2 that 5-ALA-guided surgery is more effective than conventional neuronavigation-guided surgery in increasing diagnostic accuracy and extent of tumor resection, enhancing quality of life, or prolonging survival in patients with high-grade malignant gliomas 17).
Barone et al., in a Cochrane Database Systematic Review published in 2014, that there is low to very low quality evidence (according to GRADE criteria) that image guided surgery using iMRI, 5-ALA or DTI-neuronavigation increases the proportion of patients with high grade glioma that have a complete tumour resection on post-operative MRI. There is a theoretical concern that maximising the extent of resection may lead to more frequent adverse events but this was poorly reported in the included studies. Effects of image guided surgery on survival quality of life (QoL) are unclear. Further research, including studies of ultrasound guided surgery, is needed 18).
In 2015, a literature review produced 503 potential publications; only 20 of these fulfilled the inclusion criteria of this analysis, including a total of 565 patients treated with 5-ALA-FIGR reporting on its outcomes and 800 histological samples reporting 5-ALA-FIGR sensitivity and specificity.
The mean gross total resection (GTR) rate was 75.4% (95% CI: 67.4-83.5, p<0.001). The mean time to tumor progression (TTP) was 8.1 months (95% CI: 4.7-12, p<0.001). The mean overall survival gain reported was 6.2 months (95% CI: -1-13, p<0.001). The specificity was 88.9% (95% CI: 83.9-93.9, p<0.001) and the sensitivity was 82.6% (95% CI: 73.9-91.9, p<0.001).
5-ALA-FIGR in GBM is highly sensitive and specific, and imparts significant benefits to patients in terms of improved GTR and TTP 19).
Data of 47 consecutive patients with HGG have been collected in our study (25 males, 22 females; mean age: 60.3 years, range: 27-86 years). Fluorescein (5 mg/kg of body weight) was injected intravenously right after the induction of general anesthesia. A YELLOW 560 filter was used on an OPMI Pentero 900 microscope (Carl Zeiss Meditec, Oberkochen, Germany) to complete a microsurgical tumor removal. Glioma resection and quality of life were evaluated preoperative and postoperatively.
Gross total resection (GTR) was achieved in 53.2% (n = 25) of patients. A subtotal resection (STR) (>95%) was achieved in 29.8% (n = 14), while a partial resection (PR) (<95%) was obtained in 17% (n = 8) of patients. Overall, in 83% (n = 39) of patients who underwent fluorescence-guided surgery the resection rate achieved was >95%. No adverse effects correlated to fluorescein have been recorded.
Fluorescein seems to be safe and effective in the resection of HGGs, allowing a high rate of gross total removal of contrast enhanced areas 21).
Thirty-two patients received fluorescein sodium (3 mg/kg) intravenously prior to resection. Fluorescence was intraoperatively visualized using a Zeiss Pentero surgical microscope equipped with a YELLOW 560 filter. Stereotactically localized biopsy specimens were acquired from CE and NCE regions based on preoperative MRI in conjunction with neuronavigation. The fluorescence intensity of these specimens was subjectively classified in real time with subsequent quantitative image analysis, histopathological evaluation of localized biopsy specimens, and radiological volumetric assessment of the extent of resection. RESULTS Bright fluorescence was observed in all GBMs and localized to the CE regions and portions of the NCE margins of the tumors, thus serving as a visual guide during resection. Gross-total resection (GTR) was achieved in 84% of the patients with an average resected volume of 95%, and this rate was higher among patients for whom GTR was the surgical goal (GTR achieved in 93.1% of patients, average resected volume of 99.7%). Intraoperative fluorescein staining correlated with histopathological alteration in both CE and NCE regions, with positive predictive values by subjective fluorescence evaluation greater than 96% in NCE regions.
Intraoperative administration of fluorescein provides an easily visualized marker for glioma pathology in both CE and NCE regions of GBM. These findings support the use of fluorescein as a microsurgical adjunct for guiding GBM resection to facilitate safe maximal removal 22).
A single-center, prospective, single-arm, open-label Phase II clinical trial of ALA fluorescence-guided resection of high-grade gliomas (Grade III and IV) was held over a 43-month period (August 2010 to February 2014). ALA was administered at a dose of 20 mg/kg body weight. Intraoperative biopsies from resection cavities were collected. The biopsies were graded on a 4-point scale (0 to 3) based on ALA fluorescence intensity by the surgeon and independently based on tumor cellularity by a neuropathologist. The primary outcome of interest was the correlation of ALA fluorescence intensity to tumor cellularity. The secondary outcome of interest was ALA adverse events. Sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), and Spearman correlation coefficients were calculated. RESULTS A total of 211 biopsies from 59 patients were included. Mean age was 53.3 years and 59.5% were male. The majority of biopsies were glioblastoma (GBM) (79.7%). Slightly more than half (52.5%) of all tumors were recurrent. ALA intensity of 3 correlated with presence of tumor 97.4% (PPV) of the time. However, absence of ALA fluorescence (intensity 0) correlated with the absence of tumor only 37.7% (NPV) of the time. For all tumor types, GBM, Grade III gliomas, and recurrent tumors, ALA intensity 3 correlated strongly with cellularity Grade 3; Spearman correlation coefficients ® were 0.65, 0.66, 0.65, and 0.62, respectively. The specificity and PPV of ALA intensity 3 correlating with cellularity Grade 3 ranged from 95% to 100% and 86% to 100%, respectively. In biopsies without tumor (cellularity Grade 0), 35.4% still demonstrated ALA fluorescence. Of those biopsies, 90.9% contained abnormal brain tissue, characterized by reactive astrocytes, scattered atypical cells, or inflammation, and 8.1% had normal brain. In nonfluorescent (ALA intensity 0) biopsies, 62.3% had tumor cells present. The ALA-associated complication rate among the study cohort was 3.4%.
The PPV of utilizing the most robust ALA fluorescence intensity (lava-like orange) as a predictor of tumor presence is high. However, the NPV of utilizing the absence of fluorescence as an indicator of no tumor is poor. ALA intensity is a strong predictor for degree of tumor cellularity for the most fluorescent areas but less so for lower ALA intensities. Even in the absence of tumor cells, reactive changes may lead to ALA fluorescence 23).
58 patients with high grade gliomas (°III and °IV) were included. 10 of 63 tumors (15.9 %) failed to fluoresce intraoperatively of which nine (90 %) had been adjuvantly treated prior to recurrence, as were 46 of the 53 fluorescing tumors (86.8 %). Non-fluorescing tumors were IDH mutated significantly more often (p = 0.005). 30 tumors (47.6 %) were located eloquently. 51 (80.9 %) patients showed no new neurologic deficits postoperatively. 13 patients (20.6 %) showed no signs of recurrence at their latest follow up. Eight patients were lost to follow up. Overall survival was significantly longer in the 5-ALA group (p = 0.025). Fluorescence-guided surgery in recurrent gliomas is safe and allows for a good surgical and neurological outcome in a difficult surgical environment, especially when used in combination with neuronavigation and intraoperative ultrasound to prevent over-resection. Adjuvant therapy did not significantly influence fluorescing properties 24).
Clinical and surgical data from patients affected by HGG who underwent surgery guided by 5-ALA fluorescence between June 2011 and February 2014 were retrospectively evaluated. Surgical outcome was evaluated by assessing the resection rate as gross total resection (GTR) > 98% and GTR > 90%. We finally stratified data for recurrent surgery, tumor location, tumor size, and tumor grade (IV versus III grade sec. WHO).
94 patients were finally enrolled. Overall GTR > 98% and GTR > 90% was achieved in 93% and 100% of patients. Extent of resection (GTR > 98%) was dependent on tumor location, tumor grade (P < 0.05), and tumor size (P < 0.05). In 43% of patients the boundaries of fluorescent tissue exceeded those of tumoral tissue detected by neuronavigation, more frequently in larger (57%) (P < 0.01) and recurrent (60%) tumors.
5-ALA fluorescence in HGG surgery enables a GTR in 100% of cases even if selection of patients remains a main bias. Recurrent surgery, and location, size, and tumor grade can predict both the surgical outcome and the intraoperative findings 25).
Schucht et al., prospectively studied 72 consecutive patients who underwent 5-ALA-guided surgery for a glioblastoma adjacent to the corticospinal tract (CST; < 10 mm) with continuous dynamic monopolar motor mapping (short-train interstimulus interval 4.0 msec, pulse duration 500 μsec) coupled to an acoustic motor evoked potential (MEP) alarm. The extent of resection was determined based on early (< 48 hours) postoperative MRI findings. Motor function was assessed 1 day after surgery, at discharge, and at 3 months.
Five patients were excluded because of nonadherence to protocol; thus, 67 patients were evaluated. The lowest motor threshold reached during individual surgery was as follows (motor threshold, number of patients): > 20 mA, n = 8; 11-20 mA, n = 13; 6-10 mA, n = 10; 4-5 mA, n = 13; and 1-3 mA, n = 23. Motor deterioration at postsurgical Day 1 and at discharge occurred in 30% (n = 20) and 10% (n = 7) of patients, respectively. At 3 months, 3 patients (4%) had a persisting postoperative motor deficit, 2 caused by vascular injury and 1 by mechanical injury. The rates of intra- and postoperative seizures were 1% and 0%, respectively. Complete resection of enhancing tumor was achieved in 73% of patients (49/67) despite proximity to the CST.
A rather high rate of CRET can be achieved in glioblastomas in motor eloquent areas via a combination of 5-ALA for tumor identification and intraoperative mapping for distinguishing between presumed and actual motor eloquent tissues. Continuous dynamic mapping was found to be a very ergonomic technique that localizes the motor tissue early and reliably 26).
Schucht et al., selected 13 patients who had received a complete resection according to intraoperative 5-ALA induced fluorescence and CRET according to post-operative T1 contrast-enhanced MRI. The volumes of pre-operative contrast enhancing tissue, post-operative resection cavity and resected tissue were determined through shift-corrected volumetric analysis.
The mean resection cavity (29 cm(3)) was marginally smaller than the pre-operative contrast-enhancing tumor (39 cm(3), p = 0.32). However, the mean overall resection volume (84 cm(3)) was significantly larger than the pre-operative contrast-enhancing tumor (39 cm(3), p = 0.0087). This yields a mean volume of resected 5-ALA positive, but radiological non-enhancing tissue of 45 cm(3). The mean calculated rim of resected tissue surpassed pre-operative tumor diameter by 6 mm (range 0-10 mm).
Results of the current study imply that (i) the resection cavity underestimates the volume of resected tissue and (ii) 5-ALA complete resections go significantly beyond the volume of pre-operative contrast-enhancing tumor bulk on MRI, indicating that 5-ALA also stains MRI non-enhancing tumor tissue. Use of 5-ALA may thus enable extension of coalescent tumor resection beyond radiologically evident tumor. The impact of this more extended resection method on time to progression and overall survival has not been determined, and potentially puts adjacent and functionally intact tissue at risk 27).
A retrospective review found 118 consecutive patients with high-grade gliomas operated on with the use of fluorescence-guided surgery with 5-aminolevulinic acid. Within that series, the 52 patients with newly diagnosed GBM and complete resection of enhancing tumor (CRET) in early MRI were selected for analysis. They studied the influence of residual fluorescence in the surgical field on overall survival and neurological complication rate. Multivariate analysis included potential relevant factors: age, Karnofsky Performance Scale, O-methylguanine methyltransferase methylation promoter status, tumor eloquent location, preoperative tumor volume, and adjuvant therapy.
The median overall survival was 27.0 months (confidence interval = 22.4-31.6) in patients with nonresidual fluorescence (n = 25) and 17.5 months (confidence interval = 12.5-22.5) for the group with residual fluorescence (n = 27) (P = .015). The influence of residual fluorescence was maintained in the multivariate analysis with all covariables, hazard ratio = 2.5 (P = .041). The neurological complication rate was 18.5% in patients with nonresidual fluorescence and 8% for the group with residual fluorescence (P = .267).
GBM patients with CRET in early MRI and no fluorescent residual tissue had longer overall survival than patients with CRET and residual fluorescent tissue 28).
One hundred three consecutive patients underwent resection of glioblastoma from August 2008 to November 2010. Eligibility for CRET was based on the initial magnetic resonance imaging assessed by 2 reviewers. The primary end point was the number of patients with CRET and GTR. Secondary end points were volume of residual contrast-enhancing tissue and new postoperative neurological deficits.
Fifty-three patients were eligible for GTR/CRET (n = 43 newly diagnosed glioblastoma, n = 10 recurrent); 13 additional patients received surgery for GTR/CRET-ineligible glioblastoma. GTR was achieved in 96% of patients (n = 51, no residual enhancement >0.175 cm); CRET was achieved in 89% (n = 47, no residual enhancement). Postoperatively, 2 patients experienced worsening of preoperative hemianopia, 1 patient had a new mild hemiparesis, and another patient sustained sensory deficits.
Using 5-aminolevulinic acid imaging and intraoperative mapping/monitoring together leads to a high rate of CRET and an increased rate of GTR compared with the literature without increasing the rate of permanent morbidity. The combination of safety and resection-enhancing intraoperative technologies was likely to be the major drivers for this high rate of CRET/GTR 29).
322 patients aged 23-73 years with suspected malignant glioma amenable to complete resection of contrast-enhancing tumour were randomly assigned to 20 mg/kg bodyweight 5-aminolevulinic acid for fluorescence-guided resection (n=161) or to conventional microsurgery with white light (n=161). The primary endpoints were the number of patients without contrast-enhancing tumour on early MRI (ie, that obtained within 72 h after surgery) and 6-month progression-free survival as assessed by MRI. Secondary endpoints were volume of residual tumour on postoperative MRI, overall survival, neurological deficit, and toxic effects. We report the results of an interim analysis with 270 patients in the full-analysis population (139 assigned 5-aminolevulinic acid, 131 assigned white light), which excluded patients with ineligible histological and radiological findings as assessed by central reviewers who were masked as to treatment allocation; the interim analysis resulted in termination of the study as defined by the protocol. Primary and secondary endpoints were analysed by intention to treat in the full-analysis population. The study is registered at http://www.clinicaltrials.gov as NCT00241670.
Median follow-up was 35.4 months (95% CI 1.0-56.7). Contrast-enhancing tumour was resected completely in 90 (65%) of 139 patients assigned 5-aminolevulinic acid compared with 47 (36%) of 131 assigned white light (difference between groups 29% [95% CI 17-40], p<0.0001). Patients allocated 5-aminolevulinic acid had higher 6-month progression free survival than did those allocated white light (41.0% [32.8-49.2] vs 21.1% [14.0-28.2]; difference between groups 19.9% [9.1-30.7], p=0.0003, Z test). Groups did not differ in the frequency of severe adverse events or adverse events in any organ system class reported within 7 days after surgery.
Tumour fluorescence derived from 5-aminolevulinic acid enables more complete resections of contrast-enhancing tumour, leading to improved progression-free survival in patients with malignant glioma 30).
Fifty-two consecutive patients with GBM received oral doses of 5-ALA (20 mg/kg body weight) 3 hours before induction of anesthesia. Intraoperatively, tumor fluorescence was visualized using a modified operating microscope. Fluorescing tissue was removed whenever it was considered safely possible. Residual enhancement on early postoperative MR imaging was quantified and related to each patient's characteristics to determine which factors influenced resection. Survival was analyzed using the Kaplan-Meier method and multivariate analysis was performed in which the Karnofsky Performance Scale (KPS) score, residual fluorescence, patient age, and residual enhancement on MR images were considered. Intraoperatively, two fluorescence qualities were perceived: solid fluorescence generally reflected coalescent tumor, whereas vague fluorescence mostly corresponded to infiltrative tumor. Complete resection of contrast-enhancing tumor was accomplished in 33 patients (63%). Residual intraoperative tissue fluorescence left unresected for safety reasons predicted residual enhancement on MR images in 18 of the 19 remaining patients. Age, residual solid fluorescence, and absence of contrast enhancement in MR imaging were independent explanatory factors for survival, whereas the KPS score was significant only in univariate analysis. No perioperative deaths and one case of permanent morbidity were encountered.
The observations in this study indicate the usefulness of 5-ALA-induced tumor fluorescence for guiding tumor resection. The completeness of resection, as determined intraoperatively from residual tissue fluorescence, was related to postoperative MR imaging findings and to survival in patients suffering from GBM 31).