Key-words:
Fluorescence brain tumor - fluorescence-guided surgery - high-grade glioma - sodium
fluorescein
Introduction
Maximal safe resection has been shown to correlate with improvement in both progression
free and overall survival in high-grade glioma.[[1]] Surgical adjuncts that have been shown to improve extent of resection include the
use of fluorescence, intraoperative magnetic resonance imaging (MRI), and neuronavigation.[[2]] Fluorescence has the advantage of being readily available, as well as more time
and cost-efficient than intraoperative MRI, and furthermore is not subject to the
inaccuracies encountered with brain shift or registration error with neuronavigation.[[3]]
The primary fluorescence agent that has been studied in glioma is 5-aminolevulinic
acid (5-ALA). This produces fluorescent porphyrins that accumulate in glioma cells,
resulting in fluorescence under blue light.[[4]] The limitations of this agent include cost, the need to switch between blue light
(for identification of fluorescent tissue) and white light (to delineate the anatomy
of the nonfluorescent tissue and vessels for coagulation) frequently during surgery,
as well as the side effects associated with photosensitivity of the 5-ALA compound.[[3]]
Sodium fluorescein has been suggested as an alternative to 5-ALA. Intravenous sodium
fluorescein administration results in green fluorescence under yellow light in areas
of blood–brain barrier impairment due to the accumulation of sodium fluorescein in
the extracellular space.[[3]],[[5]],[[6]] The primary criticism regarding the use of sodium fluorescein pertains to the fact
that fluorescence is not tumor cell specific – there is a potential for false-positive
fluorescence garnered by accumulation in areas of perilesional edema and surgical
tissue injury.[[7]],[[8]]
At present, there are few reports regarding the efficacy of sodium fluorescein at
tumor margins. Investigation of the specificity of sodium fluorescein in detecting
residual tumor at resection cavity margins has been largely limited to biopsy-based
histological studies in high-grade glioma patients.[[3]],[[5]],[[9]],[[10]],[[11]],[[12]],[[13]] In this study, we present our early experience with the use of sodium fluorescein
in brain tumor surgery and describe a technique that may facilitate the correlation
between marginal sodium fluorescein uptake and residual contrast-enhancing tumor.
Methods
Ethics approval was obtained through the Health District Human Research Ethics Committee,
and a clinical trial notification was provided to the Therapeutic Goods Administration
given the off-label use of the drug. Participants over the age of 18 years who were
undergoing surgery for primary or secondary brain tumors at a single university-affiliated
tertiary institution (Royal North Shore Hospital, Sydney, Australia) were invited
to participate. Patients with extrinsic tumors, nonenhancing tumors, or with hypersensitivity
to sodium fluorescein were excluded from the study. Written informed consent was obtained
in all cases.
The dosing protocol of Acerbi et al. in the FLUOGLIO study was replicated.[[3]] A single intravenous dose of 5 mg/kg of sodium fluorescein (Retinofluor, Phebra
Pty Ltd.) was administered immediately on induction of general anesthesia. Standard
white light surgical resection of the tumor was performed with the aid of neuronavigation
and the Zeiss KINEVO microscope (Carl Zeiss Meditec, Oberkochen, Germany). Fluorescence
was not used to guide further surgical resection in this study.
Intraoperative photographs of tumors and perilesional tissues were taken under white
light and with the Yellow 560 nm filter applied (Carl Zeiss Meditec, Oberkochen, Germany).
Time from administration of fluorescein to early and final tumor cavity photographs
was recorded. Tumor histology was recorded, and those patients with high-grade gliomas
underwent a postoperative MRI within 48 h of surgery to quantify the extent of resection.
A gross-total resection was defined as a >90% resection of contrast-enhancing tumor.
Residual marginal fluorescence following tumor removal was recorded through intraoperative
photography with both white light and under the Yellow 560 nm filter. In order to
distinguish areas of more avid fluorescence expression from general background fluorescence,
an open-source biological-image analysis program (FIJI)[[14]] was then used to segment the original color Yellow 560 nm filtered photograph into
red/green/blue monochrome channels. Following this, a thermal filter was applied to
the green pixel substrate to aid in identifying the areas of relatively avid green
fluorescence [[Figure 1]]. On these “thermal maps,” blue was interpreted as no fluorescence, green as mild-moderate,
and red as avid fluorescence.
Figure 1: (a) White light photograph of a well-demarcated superficial lesion. (b) Heterogeneous
fluorescence in lesion and dura demonstrated with Yellow 560 nm filter applied. (c)
Green monochrome of fluorescence photograph. (d) Thermal filter applied to green monochrome
to demonstrate moderate fluorescence intensity in the area of tumor adjacent to the
cortex
This “fluorescence map” was then analyzed by the investigators to determine which
surfaces of the final resection cavity featured ongoing high-intensity fluorescence.
Those surfaces were then recorded and explored for the presence of residual contrast-enhancing
disease on the postoperative MRI. Through this method, the sensitivity and specificity
of sodium fluorescein in detecting residual contrast-enhancing disease at tumor margins
was assessed.
The primary outcome was the specificity of marginal fluorescence in indicating the
presence of residual contrast-enhancing tumor. Secondary outcomes were dynamic changes
in intraoperative fluorescence and toxicity.
Results
Over a 3-month period (December 2018–February 2019), 11 patients with a total of 12
lesions were enrolled in the study. The mean age was 63.5 years (range: 36–74), with
a male-to-female ratio of 1:1.2. The majority of the lesions were supratentorial (75%).
All patients received a sodium fluorescein dose of 5 mg/kg without any adverse effect.
The median time from injection to initial tumor exposure was 76 min and from injection
to final cavity check was 138 min.
Histopathology demonstrated glioblastoma in seven patients (64%), metastasis in three
patients (27%), and pilocytic astrocytoma in one patient. One patient had two metastatic
lesions excised in the same operation. Three of the 11 patients were a recurrence
of a previously excised lesion (27%). Demographic data are presented in [[Table 1]].
Table 1: Demographic characteristics
Of the seven patients with glioblastoma, an early postoperative MRI was obtained in
six patients. This demonstrated gross-total resection in four patients (67%). All
four metastatic lesions were excised in an en bloc manner and deemed to be completely
excised by the operating surgeon.
All tumors in our study demonstrated fluorescence under the Yellow 560 nm filter (100%
sensitivity). As previously reported in the literature, the fluorescence characteristics
of these tumors were heterogeneous with less avid enhancement noted in areas of central
necrosis.[[3]],[[12]] Where lesions presented to the cortical surface or were immediately subcortical,
fluorescence borders correlated strongly with neuronavigation and white light assessment
of abnormal tissue [[Figure 2]].
Figure 2: White light (left) and Yellow 560 nm (right) photographs demonstrating cortical and
subcortical lesions with well-demarked superficial fluorescence boundaries
Some degree of perilesional fluorescence was seen in all cases. In particular, the
three patients that were being treated for a recurrence of a previously excised lesion
(pilocytic astrocytoma, glioblastoma, and metastasis) had marked fluorescence of the
gliotic brain around the tumor cavity [[Figure 3]]. Interestingly, all three metastatic lesions also had notable homogenous tumor
cavity fluorescence following en bloc resection. Intraoperatively, this was able to
be distinguished from the more avid fluorescence of the lesion itself. No dynamic
changes in the presence or intensity of marginal fluorescence were noted intraoperatively
or on postoperative review of intraoperating images.
Figure 3: White light (left) and Yellow 560 nm (right) photographs of two patients with recurrent
tumors demonstrating widespread sodium fluorescence uptake in tumor as well as the
surrounding gliotic brain
Peripheral fluorescence was more heterogeneous in high-grade glioma patients. In all
six glioblastoma patients who had an early postoperative MRI, analysis of tumor cavity
photographs taken with the Yellow 560 nm filter and aided by fluorescence intensity
mapping demonstrated a strong correlation between areas of relative high fluorescence
and the presence of residual contrast-enhancing tumor. Furthermore, areas of relative
quiescence on fluorescence intensity-mapped photographs were associated with an absence
of residual contrast-enhancing disease on MRI [[Figure 4]].
Figure 4: Cavity fluorescence in high-grade glioma patients. Orientation A - anterior, P -
posterior, S - superior, I - inferior, M - medial, L - lateral. (a) Strong correlation
between area of high relative fluorescence on thermal map (black arrow) and residual
tumor on magnetic resonance imaging (white arrow). (b) Redo case with high relative
fluorescence in surrounding gliotic cortex (under P) as well as medial avid fluorescence
(black arrow) which correlates with residual disease on magnetic resonance imaging
(white arrow). Residual tumor at posterior margin not obvious given more avid signal
from other areas. (c) Posterior avid fluorescence (black arrow) with correlating with
small area of posterior enhancement on magnetic resonance imaging (white arrow). (d)
No avid enhancement on thermal imaging and no residual disease on magnetic resonance
imaging. (e) Low-avidity signal from posterior cavity not reflecting of large posterior
residual on magnetic resonance imaging (white arrow). (f) High relative fluorescence
anteriorly (black arrow) correlating with small volume of residual enhancement on
subtracted magnetic resonance imaging (white arrow)
Twenty margins were assessed for residual fluorescence in the six glioblastoma patients
who had an early postoperative MRI. The presence of avid fluorescence (red on thermal
maps) had a sensitivity of 66.7% and specificity of 75% for the presence of residual
contrast-enhancing tumor on postoperative MRI. A false-negative result was recorded
in patient B and patient E with lack of high-intensity fluorescence at the posterior
cavity margin despite the presence of bulky residual disease on MRI. There was one
false positive in patient F where appropriate avid tumor fluorescence was depicted
anteriorly, but inappropriate avid fluorescence was present medially without residual
contrast-enhancing disease here on MRI.
Discussion
The use of intraoperative fluorescence demonstrates great potential in facilitating
maximal resection of tumors. 5-ALA has been shown in a Phase III study to significantly
improve the extent of resection when compared to conventional white light microsurgery.
This afforded a progression-free survival benefit in this group.[[4]] However, it has a number of limitations including cost, photosensitivity, loss
of anatomical detail under blue light, and requirement for early dosing.
Sodium fluorescein has been proposed as an alternative fluorescence agent that has
the advantages of significantly lower cost, ability to visualize anatomical detail
under yellow light, relative simplicity of dosing, and a lower adverse effect profile.
However, unlike 5-ALA which results in the accumulation of fluorescent porphyrins
in malignant glioma cells, sodium fluorescein produces nonspecific fluorescence of
areas where there is an impairment of the blood–brain barrier.[[6]]
Murray first reported on the sensitivity and specificity of sodium fluorescein in
1982.[[15]] In the past 5 years, six studies have reported on the sensitivity and specificity
of sodium fluorescein in glioma surgery.[[3]],[[5]],[[9]],[[10]],[[11]],[[12]],[[13]] In the Phase II FlUOGLIO study, Acerbi et al. found sodium fluorescein to have
a sensitivity of 80.8% and specificity of 79.1% through histopathological analysis
of fifty biopsies of fluorescent and nonfluorescent tissue at tumor margins.[[3]] Other authors also document sensitivities and specificities in excess of 80% [[Table 2]].
Table 2: Studies reporting sensitivity and specificity of sodium fluorescein
However, these results must be interpreted with a degree of caution as some authors
incorporate biopsies taken from fluorescent and nonfluorescent tissues within the
tumor core into their specificity calculation.[[10]],[[12]] These biopsies from known contrast-enhancing areas reflect the variable fluorescence
seen with necrosis in the tumor core but fail to address the key clinical question
of the significance of ongoing fluorescence in noncontrast-enhancing areas at tumor
margins.
The most rigorous analysis of residual fluorescence at tumor margins has been performed
by Neira et al.[[9]] In their study of 32 patients with glioblastoma, fluorescence always resulted in
a histopathologically abnormal biopsy in contrast-enhancing regions of tumor. They
reported an overall sensitivity of 75.6% and specificity of 75% across both contrast-enhancing
and noncontrast-enhancing regions. However, when this was limited to assessment of
residual fluorescence in the noncontrast-enhancing tumor margin, the sensitivity fell
to 69.4% and specificity to 66.7%.
Through postoperative quantification of the degree of fluorescence, they were able
to identify a “threshold value” of 0.1 normalized fluorescence intensity in only yielding
biopsies specific for tumor or infiltrating tumor in noncontrast-enhancing areas at
tumor margins.[[9]]
While Neira et al. report a valuable guide to identifying infiltrating tumor in nonenhancing
regions, their method of quantification of fluorescence requires the selection of
active and background regions of interest to determine specific quantitative fluorescence
intensity. This may be difficult and time-consuming to do in real time in the intraoperative
setting, rendering their technique somewhat impractical. Furthermore, their results
also suggest that the correlation between subjective and objective classifications
of fluorescence intensity is only strong when fluorescence is absent or high and prone
to error when fluorescence intensity is medium or low.
Our method uses a similar approach to Neira et al. in isolating the green pixel monochrome
but then utilizes a “thermal look-up table” to provide a visual display of relative
fluorescence intensity without the need for manual selection of regions of interest.
This allows quick identification of areas of intense fluorescence expression that
may not be as obvious under Yellow 560 nm light given background fluorescence. In
our study, areas of high relative fluorescence intensity correlated strongly with
the presence of residual contrast-enhancing tumor on postoperative MRI [[Figure 4]]. It is feasible that the existing microscope software could be updated to include
the ability to analyze relative fluorescence intensity in real time through overlay
of a graphical representation of fluorescence intensity onto anatomical detail in
intraoperative photographs.
The specificity of our technique (75%) matched that described by other authors who
focused their assessment on fluorescence at tumor margins.[[3]],[[9]] The sensitivity calculated by our technique (66.7%) was lower than that reported
by groups in the literature with the exception of the only other group to apply quantitative
analysis.[[9]] In our study, accurate calculation of sensitivity at tumor margins may have been
confounded by the presence of more bulky residual disease in the two patients who
recorded a false-negative margin – the lack of fluorescence may be attributable to
the presence of ongoing necrotic tumor at this margin with an expected paucity of
fluorescence expression. Although these figures are in keeping with those reported
by other groups in the literature, they are not statistically robust due to the small
sample size, lack of histopathological validation, and nonblinded assessment of postoperative
imaging and thermal maps. If integrated into the microscope software in the future,
this technique requires further validation with a combined postoperative imaging and
intraoperative biopsy-based calculation of the sensitivity and specificity of areas
of high relative fluorescence intensity for residual tumor.
A number of minor limitations regarding the use of sodium fluorescein were flagged
in our early experience. Dural fluorescence was seen in all cases but did not interfere
with assessment of fluorescence in the brain parenchyma. In some cases, we noted pooling
of the fluorescence agent in blood at the surgical site and extradurally. There was
no significant time difference between injection and assessment in these cases when
compared to other cases in which this was not observed. The utility of fluorescein
in detecting residual tumor at margins in redo surgical cases is limited by the avid
fluorescence of the surrounding gliotic brain.
It is unclear why there is perilesional fluorescence in brain metastases. This was
noted in all of our metastasis resections as well as by previous authors in a large
study of patients with brain metastases.[[16]] Subjectively, residual marginal fluorescence was felt to be homogeneous and of
lower intensity than that in the metastasis itself. Sequential intraoperative photographs
in cerebral metastasis patients did not demonstrate dynamic changes in marginal fluorescence
suggestive of fluorescence due to surgical tissue injury.
It is difficult to imagine that a histopathological study of fluorescence specificity
in these patients will yield similar results to that in patients with infiltrating
gliomas. A method of assessing relative fluorescence intraoperatively may be useful
in differentiating small areas of residual tumor from background fluorescence in these
cases. Our method of graphically depicting relative fluorescence intensity is one
such way of doing this and further study with histopathological correlation would
be the next stage in the validation of this technique.
Conclusion
This is a pilot study of the authors' initial experience with sodium fluorescein and
suffers a number of limitations including small population size, a nonhomogeneous
patient population, lack of histopathological analysis, and nonblinded assessment
of postoperative imaging and thermal maps. Although sodium fluorescein was felt to
be useful in delineating residual disease, maximal fluorescence-guided resection was
not pursued in this study due to our uncertainty regarding the sensitivity and specificity
of sodium fluorescein at tumor margins and our awareness for the potential for false-positive
fluorescence at tumor margins. The sensitivity of sodium fluorescein at tumor margins
is likely to be greater than what was reported in our study given confounding by the
presence of necrotic tumor layers on postoperative imaging.
These limitations preclude rigorous statistical analysis; however, we have been able
to replicate the success of other authors in the safe use of sodium fluorescein at
low doses and more significantly describe a potential method through which the specificity
of sodium fluorescein in detecting residual disease at tumor margins may be amplified.
The integration of this technique into the microscope software interface would permit
real time use and facilitate histopathological validation in future studies.
