Research Article Fluorescence-Guided Resection of Malignant Glioma with 5-ALA

12 Pages·2016·2.83 MB·English

Hindawi Publishing Corporation
International Journal of Biomedical Imaging
Volume 2016, Article ID 6135293, 11 pages
http://dx.doi.org/10.1155/2016/6135293
Research Article
Fluorescence-Guided Resection of Malignant Glioma with 5-ALA
SadahiroKaneko1andSadaoKaneko2
1DepartmentofNeurosurgery,TomakomaiCityHospital,No.5-20,1-chome,Shimizutyou,Tomakomai,Hokkaido053-0034,Japan
2DepartmentofNeurosurgery,KashiwabaNeurosurgicalHospital,No.7-20,15-chome,1-jyou,Tsukisamuhigashi,
Toyohira-ku,Sapporo,Hokkaido062-5813,Japan
CorrespondenceshouldbeaddressedtoSadaoKaneko;[email protected]
Received17February2016;Revised31May2016;Accepted1June2016
AcademicEditor:YantianZhang
Copyright©2016S.KanekoandS.Kaneko.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Malignantgliomasareextremelydifficulttotreatwithnospecificcurativetreatment.Ontheotherhand,photodynamicmedicine
represents a promising technique for neurosurgeons in the treatment of malignant glioma. The resection rate of malignant
glioma has increased from 40% to 80% owing to 5-aminolevulinic acid-photodynamic diagnosis (ALA-PDD). Furthermore,
ALAisveryusefulbecauseithasnoseriouscomplications.Basedonpreviousresearch,itisapparentthatprotoporphyrinIX
(PpIX)accumulatesabundantlyinmalignantgliomatissuesafterALAadministration.Moreover,itisevidentthatthemechanism
underlyingPpIXaccumulationinmalignantgliomatissuesinvolvesanabnormalityinporphyrin-hememetabolism,specifically
decreasedferrochelataseenzymeactivity.Duringresectionsurgery,themacroscopicfluorescenceofPpIXtothenakedeyeismore
sensitivethanmagneticresonanceimaging,andthealertrealtimespectrumofPpIXisthemostsensitivemethod.Inthefuture,
chemotherapywithnewanticanceragents,immunotherapy,andnewmethodsofradiotherapyandgenetherapywillbedeveloped;
however,ALAwillplayakeyroleinmalignantgliomatreatmentbeforethedevelopmentofthesenewtreatments.Inthispaper,we
provideanoverviewandpresenttheresultsofourclinicalresearchonALA-PDD.
1.Introduction survivalratewas3.8%[1].Totalresectionofthetumormeans
thattheenhancingtumorshadowcompletelydisappearson
Malignantgliomasconsistofanaplasticastrocytoma(WHO magneticresonanceimaging(MRI)aftersurgery.
grade III) and glioblastoma (WHO grade IV) and pos-
On MRI, malignant gliomas are well-circumscribed
sess a lethal prognosis. Despite surgery, radiotherapy, and
tumorswithregionalnecrosissurroundedbyviableinvading
chemotherapy, the median survival time for a patient with
tissues and marginal contrast enhancement. However, it is
glioblastoma is only 15.0 months, and it is not much better
well known that infiltrating glioma cells exist in the brain
forpatientswithanaplasticastrocytoma[1].
adjacenttothetumor,2-3cmawayfromthemainbodyofthe
Malignant gliomas are very difficult to treat. There is
glioma,andshowcontrastenhancementonMRI.Moreover,
clearly a need for new, effective, and safe treatments for
80%–90%ofglioblastomarecurrencesoccurfromthebrain
malignantglioma.
adjacenttothetumor.MRIcanfailtodetecttheseinfiltrating
Theremotemetastasisofmalignantgliomastoextracra- glioma cells in the brain adjacent to the tumor. In these
nialorgansisrare,andtherapeuticefficacydependsonlocal respects,malignantgliomashavefeaturesthatareinstriking
controlofthemalignantgliomainthebrain. contrasttoothermalignanttumorselsewhereinthebody.
Ontheotherhand,theresectionrateofgliomaisbelieved Extensiveresectionofmalignantglioma,includingnor-
to affect patient prognosis. The Committee of Brain Tumor mal tissue, has to be limited to prevent the possibility of
Registry of Japan reported on the relationship between the brain dysfunction after surgery. This is especially true for
resectionrateofmalignantgliomaandpatientsurvivaltime. tumors located in the eloquent brain areas of the speech
Aftertotalresectionofthetumor,the5-yearsurvivalratewas or motor functional centers of the brain in which serious
20.4%;however,afterresecting50%ofthetumor,the5-year complications, such as paralysis or speech disturbance, can
2 InternationalJournalofBiomedicalImaging
Glycine+succinyl CoA
ALA synthase
ALA (Feedback)
ALA dehydratase
Porphobilinogen
Heme
Porphobilinogen deaminase
Ferrochelatase+iron
Hydroxymethylbilane
Uroporphyrinogen III Protoporphyrin IX
synthase
Uroporphyrinogen III
Protoporphyrinogen
Coproporphyrinogen I oxidase
Protoporphyrinogen IX
Pentacarboxyl porphyrinogen
COPRO I Uroporphyrinogen
decarboxylase
Coproporphyrinogen III Harderoporphyrinogen
Coproporphyrinogen
oxidase
PENTA
COPRO III HARDERO
Figure 1: The simplified synthetic pathway of ALA-induced PpIX. ALA is an endogenous substance. It is incorporated into the
mitochondria, repeatedly transported between the cytoplasm and the mitochondria, and finally used for heme synthesis. ALA is
convertedintoporphobilinogenbytheenzymeALAdehydratase,andporphobilinogenissequentiallyconvertedintohydroxymethylbilane,
uroporphyrinogenIII,coproporphyrinogenIII,protoporphyrinogenIX,andfinallyPpIXbytheenzymeprotoporphyrinogenoxidase.The
finalproducthemeissynthesizedfromPpIXandiron(Fe)bytheenzymeferrochelatase(FeC).Thisbiosyntheticpathwayisconsideredtobe
thesameinallorganisms(PENTA:pentacarboxylporphyrin,COPROIorIII:coproporphyrinIorIII,andHARDERO:harderoporphyrin).
occuraftersurgery.Duetothedifficultyofextensiveresection that the residual tumor volume is <10cc while, at the same
ofmalignantglioma,residualmalignantgliomatissueisoften time, preserving the function of the surrounding normal
observeddespitetumorresection. brain.Themainaimofsurgeryistotalremovaloftheglioma,
Lacroix et al. [2] reported on the relationship between whichisthecontrast-enhancingareaonMRI.Unfortunately,
the extent of the resection and the survival time in 416 thisgoalisachievedin<35%ofcases.Therefore,itisvisually
patientswithglioblastoma.Asignificantsurvivaladvantage verydifficulttodifferentiategliomatissuefromnormaltissue
wasassociatedwithresectionof>98%ofthetumorvolume basedonitscolororhardness[4].
(median survival: 13 months) compared with resection of 5-Aminolevulinic acid (ALA) appears to be a new and
<98% of the tumor volume (median survival: 8.8 months, promising material in the field of malignant glioma treat-
𝑝 < 0.0001). In addition, Keles et al. [3] reported on ment. Because ALA is specifically taken up by malignant
the relationship between the volume of the residual tumor gliomacellsandusedforthebiosynthesisofprotoporphyrin
tissueandthevitalprognosisafterremovingthebraintumor IX (PpIX) (Figure 1), there is an abundant and specific
in 107 glioblastoma patients. It was found that the median accumulationofPpIXinmalignantgliomacells.Thetumor
survivaltimeforpatientswithcompletedisappearanceofthe fluorescence by excitation of violet-blue light is useful for
tumorshadow,asassessedbyCTorMRI,was93weeks.The photodynamic diagnosis (PDD) of the glioma at the time
mediansurvivaltimeinpatientswith10,10–20,and>20cc of surgery. ALA-PDD can distinguish the infiltrating area,
ofresidualtumorvolumewas68.7,49.0,and50.8weeks(𝑝< which is very difficult to distinguish using gadolinium-
0.0001), respectively. Furthermore, they observed that the enhanced MRI (Gd-MRI), and the following treatment is
volumeoftheresidualtumorgreatlyinfluencedthetime-to- now possible. An exclusive removal of the glioma without
recurrenceofthetumor.Theseresultsindicatethatincreasing functionaldamagetothebrainispossiblebyintraoperative
the resection rate of gliomas prolongs the patient survival PDD using ALA through distinguishing malignant glioma
time. Therefore, to ensure the best prognosis after surgical tissue from normal brain tissue with the naked eye during
removalinpatientswithmalignantgliomas,neurosurgeons surgery. This technique is known as fluorescence-guided
shouldaimtoremove>98%ofthetumorandatleastensure resectionoftheglioma[5].
InternationalJournalofBiomedicalImaging 3
2.MaterialsandMethods 2.5.4.ObservationoftheFluorescenceIntensityandSpectrum
duringSurgery. Thethreefactorsthatinfluencefluorescence
2.1. Patients. A total of 217 biopsy specimens in 125 malig- observation are distance, angle, and shading. All of these
nant glioma patients were obtained in both fluorescent factors should be kept in mind when performing photoil-
and nonfluorescent tissue. To clarify the relationship of the lumination(Figure4(a)). Fluorescence, which is difficult to
malignancy and the amount of PpIX, we obtained WHO observe macroscopically, can be confirmed by simultane-
gradeofeachgliomatissue,whichwasconfirmedbypathol- ously measuring the fluorescence spectrum. The presence
ogist. All patients provided written informed consent, and or absence of fluorescence is determined by analyzing the
the study was approved by the ethics committees of our fluorescence spectrum R/G ratio. The R/G ratio shows the
hospital. ratio of the amplitude of the red PpIX fluorescence from
the glioma tissues and the green autofluorescence from the
2.2.HighPerformanceLiquidChromatography. Theamount gliomatissuesinaspectrum(Figure4(b)).
of >25 types of intracellular trace elements and iron (Fe++)
wasmeasuredusingKondo’smethod[6].
3.Results
2.3. Pathological Examination. Histopathological examina-
3.1.PorphyrinMetabolismandPpIXContentin
tion was performed using hematoxylin and eosin (HE)
MalignantGliomaCells
staining.CellularproliferationwasassessedusingtheMIB-
1indexbyimmunohistochemistry.
3.1.1.HistologicalExamination. Fortheanalysisoftheintra-
cellulartraceelements,atotalof42tissuesampleswereused
2.4.StatisticalAnalysis. The𝜒2testwasusedtocomparethe in the biopsy specimens. In 28 of the 42 tissue samples,
distribution of the categorical variables. In all analyses, a 𝑝 glioblastoma was histologically confirmed and fluorescence
valueof<0.05(two-sided)wasconsideredtobestatistically wasobserved.In14ofthe42tissuesamples,notumorcells
significant.Allexperimentswereconductedmorethanthree and no fluorescence were observed, and they were used as
timeswithsimilarresults. controlsinthefollowingresults.
2.5.Treatment 3.1.2.IstheSubstanceEmittingtheFluorescencePpIX? Inthe
humanglioblastomatissueandcontrolbraintissuesamples,
2.5.1. Dosage and Timing of ALA Administration. 20mg/kg
the amounts of each porphyrin and the relevant enzyme
ALA was dissolved in 50mL of 5% glucose solution and
activities of porphyrin metabolism were measured. The
orallyadministeredtothepatient,approximately1hpriorto
amountofallporphyrinswasincreasedintheglioblastomas
theinductionofanesthesia.Thechemicalformulaof5-ALA
that emitted strong fluorescence after ALA administration
(5-aminolevulinicacidhydrochloride,CosmoBioCo.,Ltd.,
compared with control brain tissue without fluorescence.
Tokyo,Japan)isC2H9NO3–HCl,anditsmolecularweightis On average, the amounts of coproporphyrin and hardero-
167.59.
porphyrin increased by approximately twofold to ninefold
and that of PpIX by sixfold to ninefold. PpIX was in the
2.5.2. Light Protection (Photosensitization of the Skin). It is greatest quantity for these porphyrins at more than 10
wellknownthatphotosensitizationoftheskinlastsapprox- times (Figure 2). Therefore, we can definitely say that the
imately 24h after ALA administration. In our practice of substance emitting the fluorescence is PpIX. Harada et al.
using ALA, direct exposure of the patient to sunlight or [7] reported that PpIX shows a very strong absorbance
especially strong room light must be avoided; however, no bandatawavelengthofapproximately405nmwave(Soret
special care is needed for the patient in general indoor band) and four absorbance bands between the wavelengths
illumination. However, during or after surgery, when an of 480 and 650nm (Q bands). That is, if tissues containing
oximeterusingLEDisattachedtothepatient,caremustbe PpIX are excited using violet-blue light at a wavelength of
takentoavoidburnsfrompartoftheLEDlighttouchingthe approximately 405nm, a strong red fluorescence band at a
skin. wavelengthof635nmisobservedinthetissue.Althoughnor-
maltissuesalsosynthesizePpIX,fluorescenceisnotobserved
2.5.3. Light Illumination. Violet-blue light was used as the macroscopicallyinthesetissuesbecausetheamountofPpIX
excitationlightbecauseitirradiatesatthewavelengthofthe is very small. After ALA administration, PpIX synthesis
Soret band at which PpIX shows very strong absorbance. is observed; however, PpIX accumulation is not observed
The surgical field was irradiated with violet-blue light from in normal brain tissue. In malignant glioma tissues, PpIX
axenonlamp(Superlux301,CarlZeissJapan,Tokyo,Japan) synthesis and accumulation are observed. This means that,
throughanopticalfiberthatwasequippedwithafilter(Nikon basedontheamountofaccumulatedPpIX,differentiationof
EX405/10,400–410nm,NikonCo.,Tokyo,Japan)atthedistal malignanttumortissuefromnormalbraintissueispossible.
tip. It is possible to use other excitation light sources such PpIX emits red fluorescence after excitation with violet-
asVLD-M1(405nmDiodeLaserSystem,M&MCo.,Tokyo, blue light, and this fluorescence can be macroscopically
Japan)andsurgicalmicroscopes(OPMIPentero,CarlZeiss recognized. Therefore, tissues with red fluorescence that
Japan)equippedwithanexcitationlightsourceapparatus. contain large amounts of PpIX are very likely to be tumor
4 InternationalJournalofBiomedicalImaging
1100 ∗ inhibits the FeC activity in the malignant glioma cells. The
1000 amountsofFeinthemalignantgliomaandthecontrolswere
65.78𝜇g/gand92.89𝜇g/gofwetbrain,respectively.Although
900
thiswasnotstatisticallysignificant,theamountofFeinthe
800 malignantgliomawasobviouslysmallerthaninthecontrols.
n) 700 Theamountofhemeinthemitochondriawassmallerinthe
ai malignant glioma cells than in the control cells. This was
br 600
et thought to be because heme was not biosynthesized from
g/w 500 PpIX,althoughalargeamountofPpIXwaspresent,dueto
ng/ 400 asmallamountofFeandlowFeCactivityinthemalignant
(
300 ∗ gliomacells(Figure3(c)).Basedontheseresults,thereason
∗ ∗ whyPpIXisselectivelyaccumulatedinmalignantgliomacells
200 ∗ canbeexplainedonthebasisofporphyrin-hememetabolism
100 as follows. A large amount of PpIX is first biosynthesized
in the mitochondria, and the excessive amount of PpIX
0
PENTA COPRO I COPRO III HARDERO PpIX in the mitochondria decreases the activity of FeC. Because
the amount of Fe is small, the biosynthesis from PpIX to
Control heme decreases. Consequently, the amount of heme in the
Glioblastoma mitochondria is smaller in the malignant glioma cell than
in the control cell. We also speculated that FeC activity is
Figure2:Theamountofporphyrinsinthecontrolbraintissueand
glioblastomatissue.Thewhitebarshowscontroltissuefromabrain decreased due to the activation of PBGD and the existence
tumorpatient(𝑛 = 14),andthegraybarshowsglioblastoma(𝑛 = ofnitricmonoxide.
28)(∗𝑝<0.05)(PENTA:pentacarboxylporphyrin,COPROIorIII: The other explanation as to why ALA or PpIX is
coproporphyrinIorIII,HARDERO:harderoporphyrin,andPpIX: oncotropic is speculated to be as follows. Since porphyrins
protoporphyrinIX). haveahighaffinitytolipoprotein,especiallyto low-density
lipoprotein, which is abundant in tumor cells, PpIX, which
isaporphyrin,accumulatesinthetumorcellsbybindingto
low-densitylipoproteins.However,thereisnoevidencethat
tissue, while those without red fluorescence that contain
PpIXhasahighaffinitytolow-densitylipoprotein.
little PpIX are probably normal tissue. Many studies have
Thepept-1andpept-2transportercontrolscellularALA
addressedthequestionastowhyalargeamountoftheALA
uptake. In addition, the ATP-binding cassette transporter
metabolitePpIXisspecificallyincorporatedandaccumulated
ABCG2 plays a critical role in regulating the cellular accu-
inmalignantgliomacells.
mulationofporphyrin.
3.1.3. Why Does PpIX Excessively Accumulate in Malignant
3.1.4. Intracellular Localization of PpIX. We confirmed that
Glioma Cells? After ALA administration, ALA dehydratase
PpIXfluorescenceoccurredintissueswithhighcelldensity
obviously increased and accelerated porphyrin metabolism
and in the cytoplasm of tumor cells by comparing the
from ALA along with ALA incorporation into malignant
findings of HE stained, isolated preparations and confocal
glioma cells. It is also obvious that the material that was
laserfluorescencemicroscopy[8].
excessivelyproducedandaccumulatedwasPpIX.Toexamine
the mechanism underlying the increase in the amount of
PpIX, the activities of three enzymes, ALA dehydratase, 3.2.PracticalAspectsofALA-PDD
porphobilinogen deaminase (PBGD), and FeC, which are
involvedinporphyrinmetabolismweresimultaneouslymea- 3.2.1. Diagnostic Accuracy. On histopathology, the very
sured. In the malignant glioma cells, a decreasing trend in stronglyfluorescent tissues were activetumorcells, and the
these enzymes with increasing amounts of porphyrin was weakly fluorescent tissues showed an infiltrative area at the
observed. That is, the amount of PpIX and the activity tumormargin.Thebluefluorescenttissueswerenormalbrain
of FeC were negatively correlated. The FeC activity was tissue.Mostnecroticlesionsshowedeitherveryweakorno
expressed as the amount of heme biosynthesized for PpIX fluorescence.
per unit time. The unit of FeC activity is nmol of heme Excessive accumulation occurs not only in malignant
formed/mg/organ/h.TheFeCactivitywasclearlydecreased gliomatissuesbutalsoinothertissues.Forexample,inflam-
more in the malignant glioma cells compared with control matory tissues in acne and granulation tissue accumulate
cells(Figure3(a)).TheFeCactivityandthetotalamountof PpIX because PpIX accumulation is the result of an abnor-
PpIX in the whole cell were positively correlated; however, malityinporphyrinmetabolism.
theFeCactivityandtheamountofPpIXinthemitochondria ThemajordrawbacksofPDDaretheoccurrenceoffalse
werenegativelycorrelated(Figure3(b)).Weshowedthat,in positive and false negative results. A false positive result is
themitochondria,theFeCactivitydecreasedwithanincrease defined as the presence of fluorescence in the absence of
intheaccumulatedamountofPpIX.Theseresultsindicated histological confirmationofmalignantgliomatissues. False
that the excessive presence of PpIX in the mitochondria positivity reflects the specificity of PDD. Conversely, a false
InternationalJournalofBiomedicalImaging 5
80
∗
70
h) 60
g/
m
ol/ 50
m
n
C ( 40
e
F
of 30
y
vit
cti 20
A
10
0
Control Tumor
(a)
g)
m
ng/ 2.0 g) 4.0
ell ( g/m
ole c X (n
n the wh 1.0 R=0.94 Mt-PpI 2.0 R=−0.412
X i
pI
P
0 1.0 2.0 0 1.0 2.0
Activity of FeC (nmol/mg/h) Activity of FeC (nmol/mg/h)
(b)
∗
an) 60
g
or
g
ol/
m
n
e ( 40
m
e
h
of
nt
u
o 20
m
A
0
Control Tumor
(c)
Figure3:(a)Ferrochelatase(FeC)activity.NotethattheFeCactivityclearlydecreasedmoreinthemalignantgliomacells(𝑛=28)thanin
thecontrolcells(𝑛=14)(∗𝑝<0.01).TheFeCactivityisexpressedbytheamountofhemebiosynthesizedfromPpIXperunittime.Namely,
theunitofFeCactivityisnmolofhemeformed/mg/organ/h.TheunitofPpIXisnmol/g/organ.(b)ThecorrelationbetweentheFeCactivity
andtheamountofPpIXinthemitochondriaorinthewholecell.Theleftpanelshowstherelationshipbetweenthetotalintracellularamount
ofPpIXandtheFeCactivity.TherightpanelshowstherelationshipbetweentheamountofPpIXinthemitochondriaandtheFeCactivity.
NotethattheFeCactivityandthetotalamountofPpIXwerepositivelycorrelated(𝑅=0.94).TheFeCactivityandtheamountofPpIXinthe
mitochondriawerenegativelycorrelated(𝑅 = −0.412).Namely,theFeCactivitydecreasedwithanincreaseintheaccumulatedamountof
PpIXinthemitochondria.(c)Theamountofhemeinthemitochondria.Theamountofhemeinthemitochondriawascomparedbetweenthe
controlcells(𝑛=14)andtumorcells(𝑛=28).Thisamountwasremarkablysmallerinthetumortissuethaninthenormaltissue(∗𝑝<0.01).
6 InternationalJournalofBiomedicalImaging
(A) (B)
(C) (D)
(a)
Sample spectrum, P0, dark subtracted
10,000
9,000
R/Gratio=I(635nm)/I(505nm)
8,000
y 7,000
nsit 6,000
e Red fluorescence of PpIX
e int 5,000
ativ 4,000 Green autofluorescence
Rel 3,000 of brain
2,000
1,000
0
2 1 6 5 7 0 9 3 7 5 5 0 9 4
4 1 2 2 6 2 5 3 1 8 8 9 7 9
9. 0. 0. 9. 7. 5. 1. 7. 2. 5. 8. 0. 1. 1.
9 3 6 8 1 4 7 9 2 4 6 9 1 3
3 4 4 4 5 5 5 5 6 6 6 6 7 7
Wavelength (nm)
(b)
Figure4:(a)Anintraoperativephotographofthebrainsurface.(A)Thetumorsurfaceunderxenonwhitelight.Hypervascularityisobserved;
however,thetumorboundaryisvague.(B)Byirradiatingexcitationlightofviolet-blue,redfluorescencefromthetumortissuesisobserved
andthetumorboundaryisclearlyrecognized.(C)Inthecavityaftertumorremovalunderxenonwhitelight,theresidualtumorisnotclearly
recognized.(D)Inthesameoperativefieldas(C)underexcitationlightofviolet-blue,theresidualtumorisclearlyrecognized.(b)TheR/G
ratiowithfluorescencespectrum.Weobtainedtheamplitudeofthered(R)PpIXfluorescencefromthegliomatissuesandthegreen(G)
autofluorescencefromthegliomatissuesinaspectrum.
negative result is defined as the absence of fluorescence infiltrates, and thus extirpation of the glioma, especially
despite the histological confirmation of malignant glioma recurrentmalignantglioma,requirescarefulconsideration.
tissues.FalsenegativityreflectsthesensitivityofPDD.Tissues Inourresults,thespecificitywas92.3%,andthesensitiv-
yielding false positive results are often inflammatory cell itywas86.0%(Table1).
InternationalJournalofBiomedicalImaging 7
Table1:Therelationshipbetweenthefluorescenceandthepatho- (+++)
14
logicalexamination.
12
𝑛=217 Gliomacells Nongliomacells
10
Fluorescence(+) 153 3(allofthemwerenecrosistissues) M) (++)
Fluorescence(−) 25 36 𝜇 8
Sensitivity:86.0%;specificity:92.3%. pIX ( 6
P
4 (+)
(−)
False positivity is generally due to an abnormality in 2
hememetabolismafterPpIXsynthesisfromALA.Therefore,
0
false positive results are not confined to tumor tissues. If a Control Grade II Grade III Grade IV
similarabnormalityispresentininflammatorytissues,PpIX
WHO grading
accumulatesinthesetissuesandfluorescenceisobserved.
Figure 5: The amount of PpIX accumulation in each grade of
glioma.High-grademalignantgliomacontainsahighconcentration
3.2.2. Photobleaching during Surgery. If photobleaching of ofPpIXinthetissue.Thissignof(−)∼(+++)showstheintensityof
porphyrinsbyilluminationofthemicroscopeoccursimme- fluorescencetothenakedeye.
diately, this is a very important issue for the diagnosis of
malignant glioma during surgery. Stummer et al. [5, 9, 10]
showninFigure7.Apparentfluorescencewasnotobserved
reported that, under illumination of the microscope, the
macroscopically in lesion (a) (Figure 7(a)). Histopatholog-
fluorescencedecreasedto36%in25minforviolet-bluelight
ically, lesion (a) was composed of necrotic tissue and did
and in 87min for white light excitation. In addition, they
notshowthebandat635nmthatrepresentsPpIX.Apparent
reportedthatphotobleachingwasmuchslowerthanhadbeen
strong fluorescence was observed macroscopically in lesion
anticipated. Even if fluorescence decreases in the exposed
(b) (Figure 7(b)). Histopathologically, this lesion was active
regions of the tumor, it can be refreshed by immediate
glioblastoma with abnormal nuclei and a high cell density,
removal of the superficial tissue. Therefore, we believe that
anditsMIB-1indexwas10%.Inlesion(b),averystrongpeak
photobleachingisnotsuchaseriousissueduringsurgery.
at635nmwasobserved,andtheratioofautofluorescenceto
PpIX fluorescence (R/G ratio) was 30.9. In lesion (c) (Fig-
3.2.3. The Relationship between the Malignancy and the ure7(c)),weakfluorescencewasrecognizedmacroscopically
AmountofPpIXintheTissue. Wemeasuredtheamountof andhistopathologically,andmostofthetumorwasidentified
PpIXineachgradeofglioma.Whiletheamountincontrol asalow-gradegliomainfiltratingintothenormalbraintissue.
brain tissue was 1.73𝜇M (micromole), it was 2.29𝜇M in The MIB-1 index of lesion (c) was 0.5%, and its spectrum
WHOgradeIIglioma,7.43𝜇MinWHOgradeIIIanaplastic showed a relatively strong peak at 635nm, and the R/G
astrocytoma, and 13.65𝜇M in WHO grade IV glioblastoma ratio was 6.5. In lesion (d) (Figure 7(d)), no fluorescence
(Figure5).TheamountofPpIXwas1.8timesgreateringrade wasdetectedmacroscopicallywiththenakedeye.However,
II,4.3timesgreateringradeIII,and7.9timesgreateringrade its spectrum showed a very weak peak at 635nm and the
IVcomparedwiththeconcentrationinnormalbraintissue. R/G ratio was 1.3. The tissue in lesion (d) was identified
The amount of intracellular PpIX increased exponentially histopathologically as normal brain tissue with a few infil-
withtheincreaseinmalignancy. tratinggliomacells(guerillacells).ItsMIB-1indexwas0.5%.
In lesion (e) (Figure 7(e)), no fluorescence was observed
macroscopically, and no peak was observed at 630nm. The
3.2.4. The Fluorescence Spectrum with ALA-PDD Is Most
tissue in lesion (e) was confirmed on histopathology to be
Useful for the Estimation of Tumor Size during Surgery.
normal brain tissue. The greatest diameter of the glioma
We examined the tumor size that could be recognized by
that was distinguished from the normal tissue was 35mm
histopathologicalfindings,theR/Gratiointhefluorescence
(Figure 7). In this range, the spectrum showed an obvious
spectrum, the visual fluorescence strength, and the visual
bandat635nm,anditwashistopathologicallyconfirmedas
observationunderthewhitelight.
gliomatissue.
Thiscasewasafemalewithrighttemporallobeglioblas-
These results suggest that fluorescence diagnosis during
toma. An MR image with gadolinium contrast is shown in
surgery can successfully distinguish malignant gliomas of a
Figure 6(a) and a tumor shadow is observed in the right
sizeof35mmfromnormaltissue.Furthermore,thefluores-
temporal lobe. Figures 6(c) and 6(d) show the cut surface
cence spectrum during surgery can be used for diagnosis,
of the central portion of the tumor after total resection
even if fluorescence is not observed macroscopically. As
of the right temporal lobe glioblastoma. Figure 6(c) is a
a method for diagnosing a malignant glioma from the
picture of the surface under white light and the tumor is
normalbrainduringsurgery,wecansaythatthefluorescence
recognized by its brownish color. Figure 6(d) is the same
spectrumhasexcellentdiagnosticcapability.
surfaceasFigure6(c)underexcitationviolet-bluelightand
thefluorescenceofthetumorisclearlyrecognized.
Whether the tumor could be recognized or not was 3.2.5.CurativeEffectsofALA-PDD. Weobtainedthefollow-
examinedinfivelesions(lesions(a),(b),(c),(d),and(e))as ingsurgicalresultsusingALA-PDD.Thetotalremovalrateof
8 InternationalJournalofBiomedicalImaging
24mm
(a) (b)
30mm
20mm
(c) (d)
Figure6:Thesizeofthetumorusingdifferentmethods.(a)MRIimageshowstheglioblastomaintherighttemporallobe.(b)Itwas24mm
onenhancedMRI.(c)Itwas20mmonmacroscopicobservationunderthewhitelight.(d)Itwas30mmonmacroscopicobservationand
fluorescencetothenakedeye.
theglioblastomabeforeusingALA-PDDwas40%.However, resectionsurgeryoftheglioblastomabeforeusingALA-PDD
thetotalremovalrateafterusingALA-PDDroseto78%. was 36%. However, the total removal rate after using ALA-
PDD rose to 66%. In cases of total resection of fluorescing
tissues,themeansurvivaltimewas101weeks(23.5months).
4.Discussion Incaseswheresolidfluorescingtissueswereleftunresected,
themeansurvivaltimewas51weeks(11.9months).Inaddi-
4.1. The Value of ALA-PDD for Malignant Glioma Surgery. tion,6-monthprogression-freesurvivalwas40.1%–46.0%in
Tumor resection using fluorescence of ALA-induced PpIX theGFRgroupand21.1%–28.3%inthecontrols.
does not differ greatly from conventional neurosurgical
microsurgery.
Manymethodsareusedtodistinguishtheborderofthe 5.Conclusions
malignantgliomafromthenormalbrain.Forexample,there
areneuronavigationsystems, ultrasonography,orMRI dur- PhotodynamicapplicationusingALAisanewandpromising
ingsurgery.TumorresectionusingfluorescenceofALAhas technique for neurosurgeons in the treatment of malignant
now been added to these methods and probably represents glioma.ALA-PDDhassucceededinincreasingtheresection
the best one. Although these methods can distinguish the rateofmalignantgliomafrom40%to80%.PDDsignificantly
borderofthemalignantgliomapathologically,unfortunately, prolongs the interval until recurrence. Furthermore, with
thesemethodscannotdistinguishtheregionsofbrainfunc- intraoperative PDD for identifying tumor tissues, measure-
tion.Whenmalignantgliomacellsinfiltrateintotheeloquent ment of the fluorescent spectrum facilitates more accurate
brainareathatisacenterofspeechormotorfunctionwith and extensive resection compared with macroscopic obser-
fluorescence,speechandmotorfunctionmustbemonitored vationofthefluorescence.
to avoid resection of the eloquent areas with malignant Based on previous research, it is apparent that PpIX
glioma [11]. Therefore, PDD using ALA can distinguish accumulates abundantly in malignant glioma tissues after
malignant glioma more widely than MRI. Distinguishing ALA administration. Furthermore, it is evident that the
malignantgliomausingfluorescencehassuperiorspecificity mechanism underlying PpIX accumulation in malignant
comparedwithneuronavigationsystems,ultrasonography,or glioma tissues involves an abnormality in porphyrin-heme
MRI[12]. metabolism,specificallydecreasedFeCenzymeactivity.
Stummer et al. [5, 10, 13] described the clinical curative Although chemotherapy with new anticancer agents,
effects of ALA-PDD as follows. The total removal rate by immunotherapy,andnewmethodsofradiotherapyandgene
InternationalJournalofBiomedicalImaging 9
35mm
(e)
(d)
(c)
(b)
(a)
DZE dark subtracted
60,000
55,000 R/Gratio=0.7 MIB-1index:0%
50,000
45,000
nsity 3450,,000000
e
nt 30,000
ve i 25,000
elati 20,000
R 15,000
10,000
5,000
0
951715786266377239
700600712045378600
9.5.0.4.9.3.6.0.3.6.8.0.2.3.4.5.6.6.
925792479136802468
344445555666677777
Wavelength (nm)
(a)
DZE dark subtracted
60,000
55,000 R/Gratio=30.9 MIB-1index:10%
50,000
45,000
y 40,000
nsit 35,000
e
nt 30,000
ve i 25,000
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el
R 15,000
10,000
5,000
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700600712045378600
9.5.0.4.9.3.6.0.3.6.8.0.2.3.4.5.6.6.
925792479136802468
344445555666677777
Wavelength (nm)
(b)
DZE dark subtracted
60,000
55,000 R/Gratio=6.5 MIB-1index:0.5%
50,000
45,000
y 40,000
nsit 35,000
e
nt 30,000
ve i 25,000
ati 20,000
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R 15,000
10,000
5,000
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700600712045378600
9.5.0.4.9.3.6.0.3.6.8.0.2.3.4.5.6.6.
925792479136802468
344445555666677777
Wavelength (nm)
(c)
Figure7:Continued.
10 InternationalJournalofBiomedicalImaging
DZE dark subtracted
60,000
55,000 R/Gratio=1.3 MIB-1index:0.5%
50,000
45,000
y 40,000
nsit 35,000
e
nt 30,000
ve i 25,000
ati 20,000
el
R 15,000
10,000
5,000
0
951715786266377239
700600712045378600
9.5.0.4.9.3.6.0.3.6.8.0.2.3.4.5.6.6.
925792479136802468
344445555666677777
Wavelength (nm)
(d)
DZE dark subtracted
60,000
55,000 R/Gratio=0.6 MIB-1index:0%
50,000
45,000
y 40,000
nsit 35,000
e
nt 30,000
ve i 25,000
ati 20,000
el
R 15,000
10,000
5,000
0
951715786266377239
700600712045378600
9.5.0.4.9.3.6.0.3.6.8.0.2.3.4.5.6.6.
925792479136802468
344445555666677777
Wavelength (nm)
(e)
Figure7:Theanalysisofthefluorescencespectrumandthehistologyineachlesion.Lesion(a)wascentralnecrosiswithoutmacroscopic
fluorescence.Lesion(b)showedthehighestfluorescencemacroscopically.Lesion(c)showedafaintfluorescencemacroscopically.Lesion(d),
adjacenttolesion(c),didnotshowmacroscopicfluorescence.Lesion(e)didnotshowanyfluorescence.Theleftpanelshowstheanalysisof
thefluorescencespectrum,andtherightpanelshowsthehistologicalexaminationsandMIB-1index.
therapy will be developed in the future, we believe that includingpituitaryadenomas,metastaticbraintumors,and
the curative effect of this new treatment has the potential malignantmeningiomasthathaveinvadedintotheskullbase.
to show a maximum effect by maximum cytoreduction From our clinical experience, the following advantages
with photodynamic application using ALA. Therefore, we and disadvantages are apparent. Advantages are as follows:
strongly believe that photodynamic application using ALA (1) This method was able to distinguish malignant brain
willplayakeyroleinmalignantgliomatreatmentbeforethe tumortissuesfromnormalbraintissueswiththenakedeye
developmentofnewtreatmentsinthefuture. before tumor removal at the time of removal surgery. (2)
Many ALA derivatives have been synthesized by ALA Photodynamicmedicinehasfewsideeffectsandprovidesa
research. For example, there are ALA ether, methyl-ALA, good quality of life. Disadvantages are as follows: (1) Using
andhexyl-ALA.Thesenewderivativeshavebeendeveloped PDD, it is very difficult to distinguish infiltrative tumor
because it is known that their tumor affinity is higher than tissues or guerilla cells from normal tissues. Furthermore,
ALA. Furthermore, these new derivatives may be more therearefalsenegativeandfalsepositivefindingsespecially
suitable for fluorescence detection because they appear to in edematous brain tissues or recurrent tumor tissues. (2)
induce PpIX with better tumorselectivity. Ourhopeis that Theconcentrationofthephotosensitizerinmalignantbrain
thesenewALAderivativeswillbeusedinthefuture. tumortissuesisheterogeneous.
Furthermore,ALA-PDT(photodynamictherapy)hasthe
potentialtotreatmalignantgliomasnearorattheeloquent CompetingInterests
area of the brain without major neurological deficits [14].
It is known that photodynamic application using ALA is The authors declare that there is no conflict of interests
alsoeffectiveforbraintumorsotherthanmalignantglioma, regardingthepublicationofthispaper.