morphological imaging using magnetic resonance imaging ( mri ) is the most commonly used method for obtaining tumor information .
gd - enhanced t1-weighted mri provides anatomical imaging with hyperintense neovascularization enclosed in a hypointense region of central necrosis .
in addition , fluid - attenuated ir ( flair ) detects the surrounding edema associated with infiltrating tumor cells .
the use of positron emission tomography ( pet ) , an imaging modality providing metabolic and molecular information , can improve diagnostic procedures in malignant brain tumors .
2-deoxy-2-[f ] fluoro - d - glucose ( fdg ) , a commonly used tracer for neoplasm detection , exhibits limited utility in brain tumor imaging , because the uptake is nonspecific and can occur in any region with increased metabolic activity .
in addition , fdg - pet is a marker of glycolytic metabolism and not cellular proliferation .
l - methyl - c - methionine ( met ) is a well - established pet radiotracer for brain tumor detection and tumor delineation [ 6 , 7 ] .
amino acids , including met , readily cross the intact blood - brain barrier ( bbb ) through neutral amino acid transporters and are incorporated into the area with active tumor .
it has been shown that met uptake in gliomas significantly correlates with the who tumor grade and cell proliferation determined by ki-67 index [ 4 , 911 ] .
however , increased met uptake in nonneoplastic lesions including inflammation , infarction , and hemorrhage may result in false positives [ 12 , 13 ] .
also , the short half - life of c ( 20  min ) and rapid in vivo degradation make met - pet less useful for routine clinical use .
a fluorinated thymidine analog , 3-deoxy-3-[f ] fluorothymidine ( flt ) , has emerged as a promising pet tracer for evaluating tumor - proliferating activity in various malignant brain tumors [ 3 , 14 , 15 ] .
( tk1 ) , a principle enzyme in the salvage pathway of dna synthesis , and trapped inside the cells .
the application of flt phosphorylation as a marker of cell proliferation is based on the assumption that cellular flt trapping is a representation of thymidine incorporation into dna [ 16 , 17 ] .
since flt uptake in the normal brain tissue is very low , flt - pet provides a low - background brain image , making it an ideal pet tracer for the imaging of brain tumors .
in addition , flt - pet has been utilized in the prognostic assessment and evaluation of treatment response in malignant gliomas . despite the superior features met and flt offer as glioma cell tracers ,
the cut - off value of normal versus tumor cell infiltration is difficult to determine .
therefore , the purpose of this prospective study was to clarify the individual and combined roles of fdg - pet , met - pet , and flt - pet in tumor detection , noninvasive grading , and assessment of cellular proliferation rate in 54 newly diagnosed histologically verified gliomas of different grades .
  from april 2006 through october 2011 , 54 patients with newly diagnosed gliomas ( 23 men and 31 women ; mean age : 52.0  18.1  y ; range : 2286  y ) were enrolled in this study ( table 1 ) .
the use of fdg , met , and flt as a pet tracer was approved by the kagawa university school of medicine human subjects ethics committee , and informed written consent was obtained from all patients who participated in this study .
all tumors were graded by the world health organization ( who ) grading system ( malignancy scale ) for cns tumors .
tumor types and grades were distributed as follows and listed in table 1 : who grade ii diffuse astrocytoma ( n = 9 ) , who grade ii oligoastrocytoma ( n = 2 ) , who grade ii oligodendroglioma ( n = 1 ) , who grade iii anaplastic astrocytoma ( n = 11 ) , anaplastic oligoastrocytoma ( n = 3 ) , who grade iii anaplastic oligodendroglioma ( n = 1 ) , who grade iv glioblastoma multiforme ( n = 26 ) , who grade iv gliosarcoma ( n = 1 ) .  
hematoxylin - and - eosin - stained specimens were checked to determine the histological tumor type .
the cellular proliferation activity of the tumor was determined by measuring the ki-67 proliferation index obtained by immunohistochemical staining with anti - ki-67/mib-1 antibody ( dako , tokyo , japan ) .
the percentage of tumor cells which stained positively for ki-67 antigen was measured in the area containing the largest number of positive tumor cells and was regarded as representative of the tumor proliferation activity .
pet studies were performed using an ecat exact hr + scanner and a biograph mct 64 ( siemens / cti , knoxville , tn , usa ) in three - dimensional acquisition mode .
the image system enabled the simultaneous acquisition of 51 transverses per field of view ( fov ) , with intersection spacing of 3  mm , for a total axial fov of 15  cm .
the in - plane ( transverse ) reconstructed resolution was 4.7  mm full - width at half - maximum ( fwhm ) in the brain fov .
images were reconstructed using the filtered backprojection method with a hanning filter ( kernel fwhm 10  mm , cutoff frequency 0.4 cycle / projection element ) to generate 128  128 matrices .  
pet radiotracers were produced using the hm-18 cyclotron ( sumitomo heavy industries , tokyo , japan ) .
the resultant co2 was reduced to c - methanol by lithium aluminum hydride and subsequently converted to c - ch3i by the addition of hydrogen iodide following the modified method described by ishiwata et al . .
each patient received the fdg , met , and flt one by one for a continuous 3-day course before the surgery .
for the fdg - pet study , enteral and parental sources of glucose were withheld for at least 6  h before the examination .
no special dietary instructions were given to the patients before the met and flt - pet examination .
images were acquired with patients in the supine position , resting , with their eyes closed . using ge rod sources rotating around the head , transmission images of the brain were obtained for 5  min for fdg - pet , 3  min for met - pet , and 5  min for flt - pet .
transmission scan with ge rod can be replaced to ct attenuation correction in mct64 system . a dose of 147295  mbq ( mean dose :  208  39  mbq ) of f - fdg , 113389  mbq ( mean dose :  211  65  mbq ) of c - met , or 129236  mbq ( mean dose :  161  25  mbq ) of f - flt was injected intravenously .
regional emission images of the brain were obtained for 5  min , beginning 45  min after the f - fdg injection ; for 5  min , beginning 10  min after the c - met injection ; for 10  min , beginning 40  min after the f - flt injection .
fdg , met , and flt uptake in the brain tumor were semiquantitatively assessed by evaluating the standardized uptake value ( suv ) .
a region of interest ( roi ) was set manually by an observer around the hottest area of each lesion .
the maximum value of suv ( suv max ) was regarded as the representative value of each tumor . to calculate the tumor - to - normal tissue count density ( t / n ) ratios , the roi was set on the normal brain parenchyma ( usually contralateral normal cerebral tissue excluding ventricles ) and the mean value of suv ( suv mean ) was calculated .
the t / n ratio was determined by dividing the suv max of the tumor by the suv mean of the normal brain tissue .
co - registration of fdg - pet / met - pet / flt - pet / mri was undertaken on the workstation with a commercial software package ( dr .
the software allowed an operator to perform manual intervention ; however , required adjustment for co - registration was minimal .
the measured suv max and t / n ratios of fdg , met and flt were compared with the histological diagnoses obtained by biopsy or resection .
the relationships between the ki-67 index and suv max or t / n ratio of fdg , met and flt were evaluated .
the relationship between fdg and met or flt and met uptake were evaluated . regarding the area with a high - intensity signal on the mri ( flair ) of each patient , based on a predetermined roi , the mean suvs of fdg , met and flt were used to compare the values between fdg and met , between flt and met , and between fdg and flt .
the mann - whitney u test was used to assess the statistical significance in mean volumes across modalities .
  among the 54 glioma cases , the met accumulation was observed in 51 cases ( 94.4% ) in the met - pet study and the flt accumulation was observed in 50 cases ( 92.6% ) in the flt - pet study . regarding the 3 cases without the met accumulation and the 4 cases without the flt accumulation , they were grade ii gliomas .
all malignant gliomas ( grades iii and iv ) showed accumulation in both studies . among the cases with t / n ratios higher than 1 in the fdg - pet study , there were 2 cases ( 16.7% ) of the grade ii gliomas , 8 cases ( 53.3% ) of the grade iii gliomas , and 17 cases ( 63.0% ) of the grade iv gliomas ( table 1 ) . in fdg - pet study , it is possible to differentiate gliomas by the who grade with the sensitivity of 53.3% , the specificity of 55.6% , the false positive of 44.4% , and the false negative of 46.7% . in met - pet study
, it is possible to differentiate gliomas by who grade with the sensitivity of 95.3% , the specificity of 9.1% , the false positive of 90.9% , and the false negative of 4.7% . in met - pet study
, it is possible to differentiate gliomas by who grade with the sensitivity of 93.9% , the specificity of 20% , the false positive of 80% , and the false negative of 6.1% .  
there were no significant differences in the suv and t / n ratio among each group of the different who grades in the fdg - pet study ( figures 1(a ) and 1(d ) ) .
suv of the normal brain in the met - pet study was 1.52  0.36 ( 0.922.62 ) .
regarding the tumors in the met - pet study , mean suv max was 3.04  1.56 for the grade ii gliomas , 5.22  2.09 for the grade iii gliomas and 5.12  2.44 for the grade iv gliomas ( figure 1(b ) ) .
t / n ratio was 2.03  1.02 for the grade ii gliomas , 3.67  1.38 for the grade iii gliomas and 3.76  1.78 for the grade iv gliomas ( figure 1(e ) ) .
the mean suv max and t / n ratio of the grade iv gliomas were significantly higher than that of the grade ii gliomas , while there were no significant differences between the grade ii gliomas and grade iii gliomas . in the present study
, there were some oligodendroglioma cases , which were in grade ii , but their suv max were high and ranged from 4.0 to 6.8 .
suv of the normal brain in the flt - pet study was 0.20  0.05 ( 0.130.28 ) . regarding
the tumors in the flt - pet study , mean suv max was 0.36  0.23 for the grade ii gliomas , 0.96  0.58 for the grade iii gliomas , and 2.38  1.07 for the grade iv gliomas ( figure 1(c ) ) .
the mean t / n ratio was 1.99  1.37 for the grade ii gliomas , 4.43  2.61 for the grade iii gliomas and 11.54  6.86 for the grade iv gliomas ( figure 1(f ) ) .
there were significant differences in both suv max and t / n ratio between the grade iii gliomas and the grade iv gliomas , but , similar to the met study , there were no significant differences between the grade ii gliomas and the grade iii gliomas .
when the accumulation of met or flt within the tumor and the ki-67 index ( an index for proliferation ability of tumor ) were compared , linear regression analysis revealed a significant correlation between the ki-67 index and met suv max ( r = 0.32 , p = 0.02 ) ( figure 2(b ) ) or met t / n ratio ( r = 0.39 , p < 0.01 ) ( figure 2(e ) ) , as well as flt suv max ( r = 0.73 , p < 0.001 ) ( figure 2(c ) ) or flt t / n ratio ( r = 0.63 , p < 0.001 ) ( figure 2(f ) ) .
flt , compared to met , demonstrates a significantly strong correlation with the proliferation ability .
in fdg - pet study , linear regression analysis revealed no correlation between the ki-67 index and suv max ( r = 0.16 , p = 0.24 ) ( figure 2(a ) ) or t / n ratio ( r = 0.33 , p = 0.02 ) ( figure 2(d ) ) .
regarding the area with a high intensity signal on the mri ( flair ) of each patient , based on a predetermined roi , t / n ratio of fdg , met and flt were used to compare the values between fdg and met , between flt and met and between fdg and flt . between fdg and met , the t / n ratio of the met tended to be high without correlating with increased t / n ratio of the fdg because the oligodendroglioma components were included in the grade ii or grade iii gliomas ( figures 3(a ) and 3(b ) ) .
t / n ratio of the fdg tended to be high with correlating with low t / n ratio of the met showing a tendency toward the focus of the epileptic changes ( figures 3(a ) and 3(b ) black circle ) .
a significant but weak correlation is observed between the individual t / n ratio of fdg and met in the grade iv gliomas ( r = 0.57 , p = 0.03 ) ( figure 3(c ) ) . between flt and met , the t / n ratio of the met tended to be high without correlating with increased t / n ratio of the flt because the oligodendroglioma components were included in the grade ii or grade iii gliomas ( figures 3(d ) and 3(e ) ) .
we observed the same findings on the correlation between fdg and met . on the other hand
, no significant correlation is observed between the individual t / n ratio of flt and met in the grade iv gliomas ( r = 0.13 , p = 0.61 ) ( figure 3(f ) ) .
linear regression analysis showed a significant correlation between flt and met ( r = 0.21 , p = 0.008 ) in most areas and these areas appeared as astrocytic tumors ( figure 3(f ) , red circle ) .
the t / n ratio of the met tended to be high without correlating with increased t / n ratio of the flt ( r = 0.53 , p = 0.014 ) , because the oligodendroglioma components were included in the grade iv gliomas ( figure 3(f ) , blue circle ) .
the t / n ratio of the met tended to be low without correlating with increased t / n ratio of the flt ( r = 0.12 , p = 0.11 ) because the necrotic components were included in the grade iv gliomas ( figure 3(f ) , green circle ) .
a significant correlation is observed between the individual t / n ratio of fdg and flt in the grade iv gliomas ( r = 0.54 , p = 0.05 ) . between fdg and flt , t
/ n ratio of the fdg tended to be high with correlating with low t / n ratio of the flt showing a tendency toward the focus of the epileptic changes ( figures 3(g ) and 3(h ) black circle ) .
the t / n ratio of the fdg tended to be low without correlating with increased t / n ratio of the flt , because the necrotic components were included in the grade iv gliomas ( figure 3(i ) , green circle ) .
figures 4 and 5 , respectively , show representative cases of fdg , met , and flt - pet studies in anaplastic astrocytoma and glioblastoma .
flair showed a high - intensity lesion ( figure 4(a ) ) , and gd - enhanced mri showed slightly enhanced lesion in the right frontal lobe ( figure 4(b ) ) .
fdg - pet demonstrated increased fdg uptake within the tumor ( figure 4(c ) ) .
met - pet demonstrated increased met uptake within the tumor ( figure 4(d ) ) .
flt - pet demonstrated increased flt uptake within the tumor ( figure 4(e ) ) .
areas of different enhancement and uptake between fdg , met and flt are projected on the tumor , in order to perform histological sampling for further correlation during the resection ( figure 4 yellow arrow , red arrow ) .
yellow arrow demonstrates anaplastic astrocytoma ( figure 4(f ) ) within t / n ratio of fdg - pet ( 2.85 ) , met - pet ( 4.57 ) and flt - pet ( 9.65 ) . ki-67
index from the specimen indicated by yellow arrow was 20% ( figure 4(g ) ) .
red arrow demonstrates diffuse astrocytoma area ( figure 4(h ) ) within t / n ratio of fdg - pet ( 0.76 ) , met - pet ( 2.08 ) and flt - pet ( 4.65 ) . ki-67
index from the specimen indicated by red arrow was 8% ( figure 4(i ) ) .
he experienced an onset of aphasia and gerstmann 's syndrome and developed glioblastoma which was recognized with a ring - like contrast in the left temporo - occipital lobe on the gd - enhanced mri ( figure 5(a ) ) .
the mri study did not show a uniformed contrast , and some areas had high - intensity signal while others did not .
similar to the mri study results , the accumulation was not uniform in met - pet and flt - pet studies .
this relationship between met and flt is divided into three parts , similar to the observation described in figure 3(f ) . in the blue arrow area ,
the contrast by mri was not strong , but the t / n ratio of met was 4.31 and the t / n ratio of flt was 3.24 with a significant accumulation of met . in the green arrow area ,
the contrast by mri was strong , the t / n ratio of met was 1.60 , and the t / n ratio of flt was 5.72 with a significant accumulation of flt . in the red arrow area , the contrast by mri was recognized , but the t / n ratios of met and flt were both high ( 3.27 and 5.28 ; resp . , figures 5(b ) and 5(c ) ) .
the diagnosis revealed the blue arrow area mostly with a histological picture of oligodendroglioma ( figure 5(d ) ) with a ki-67 index of 30% ( figure 5(e ) ) and the green arrow area mainly with a histological picture of necrotic tissues ( figure 5(f ) ) with a ki-67 index of less than 10% ( figure 5(g ) ) .
as for the red arrow area displaying high accumulation of both met and flt , this area showed a strong nuclear atypia with megakaryocytes with a high ki-67 index of 70% ( figures 5(h ) and 5(i ) ) . taken together , increased accumulation of both tracers ( met and flt ) appears to indicate high - grade malignancy .
accurate tissue diagnosis of glioma is important in order to determine appropriate therapeutic strategy and predict prognosis .
final diagnosis of the grade of malignancy is carried out by surgical histopathological diagnosis . however , due to the diverse nature of gliomas within tumors , some glioma cases tend to be diagnosed less stringent than actual grade of malignancy through pathological diagnosis of a specimen taken from the tumor .
the image analysis using pet has an advantage of spatial evaluation of diverse malignant gliomas [ 8 , 21 , 22 ] .
fdg has been widely used as a pet tracer for diagnosis of malignant brain tumors .
the accumulation of fdg increases in highly malignant tumors , and this method is highly specific as an image analysis and useful for predicting prognosis .
however , fdg uptake is high in the normal brain cortex where glucose metabolism is active , and the difference between the normal accumulation and brain tumors may not be detected .
in addition , fdg uptake is low in low - grade tumors , and fdg has limitations for detecting the localization of small tumors or accurately identifying tumor area [ 5 , 24 , 25 ] .
mixed neuronal and glial tumors and gliomas which easily cause convulsions had decreased accumulation of fdg until now [ 26 , 27 ] . in our case ,
accumulation of fdg was high in low - grade glioma which is frequently associated with convulsions .
it seems that additional attention is necessary regarding the convulsive cases and accumulation of fdg .
flt is a tracer of labeled thymidine for evaluating dna synthesis , and its accumulation is considered to reflect tumor cell proliferation activity [ 9 , 28 , 29 ] . in the present study ,
there were significant differences between the grade ii gliomas and the grade iv gliomas and no significant differences between the grade ii gliomas and grade iii gliomas in the met - pet study .
however , the presence of tumor cells was confirmed in the area with increased accumulation of met but poorly depicted on mri scan and no increased accumulation of tracers in the fdg - pet and flt - pet studies , indicating its usefulness as a testing modality to determine the presence of tumor . on the other hand , the correlation of met uptake in the microvessel density and blood volume in tumor or with expression of vascular endothelial cells , which are amino acid transporters ( lat1 ) in tumor cells
is suggested , and the use of met by itself seems difficult for making diagnosis since additional factors other than proliferation activity are involved [ 8 , 30 ] .
also , it has been reported that the accumulation of met is high in oligodendroglioma due to the high cell density , and special attention is required for its differentiation .   in the flt - pet study
, there were significant differences between the grade ii gliomas and the grade iv gliomas and between the grade iii gliomas and grade iv gliomas .
in addition , there was a significant correlation between the ki-67 index and the flt accumulation .
there is a correlation between the ki-67 index and the met accumulation , but the correlation with flt is more significant .
hence , we considered that flt - pet was a more ideal and attractive pet tracer for glioma imaging than met - pet .
furthermore , the uptake of flt in normal brain tissue was lower than that of met ( suv max , 0.20  0.05 versus 1.52  0.36 ) .
one of the reasons for low uptake of flt in normal brain tissue is that flt allows the direct measurement of cellular tk1 activity , which has been proportional to the proliferation activity . since , met accumulation provides an indirect measure of lat1 expression in the vascular endothelial cells and proliferation status as amino acid uptake , met - pet exhibits higher background than flt - pet in normal brain tissue
however , flt - pet had some weak points . in our series of who grade ii glioma , uptake of flt was lower than that of met ( suv max , 0.36  0.23 versus 3.04  1.56 ) and the uptake ratios for tumor to normal brain were same ( t / n ratio , 1.99  1.37 versus 2.03  1.02 ) . who grade ii gliomas had no contrast enhancement on gd - enhanced mri .
flt uptake was similar to the low levels observed in no enhanced lesions of mri .
flt may be less useful in assessing proliferation in noncontrast - enhancing tumors regardless of the histopathology grading .
low - tracer access to the tumor tissue may limit the metabolic trapping of flt after the phosphorylation even in proliferative tumors .
the leakage of flt was observed in the area with radiation necrosis and disrupted blood - brain barrier ( bbb ) that result in the increased accumulation [ 5 , 24 , 27 ] .
we will continue the analysis of flt with more patients as flt can be quantified by kinetic analysis .
specifically , the kinetic analysis of flt - pet may clarify whether the transport effect or metabolic trapping largely contribute to the increased accumulation of flt in the tumor .
as shown in figure 5 , there were differences in the accumulation of each tracer within the same tumor due to the characteristics of each tracer , which may reflect the differences in tissues by site .
in other words , we can predict that there are many oligodendroglioma components in the area with high met accumulation and low flt accumulation , and it is likely that the area is necrotic when met accumulation is low , but flt accumulation is high .
when both tracer accumulations are high , the area is highly malignant as a tumor site and has high proliferative ability .
however , it is difficult to make differential diagnosis , determine the recurrence of tumor , and determine the presence or absence of radiation necrosis using only one type of pet studies [ 31 , 32 ] .
our study indicated that the combinational uses of fdg , met , and flt - pet help us evaluate the differences in malignancy grade of the tissues and predict the evaluation of histopathological components .
we can predict that there are many oligodendroglioma components in the area with high met accumulation and low flt accumulation , and it is likely that the area is necrotic when met accumulation is low but flt accumulation is high .
when both met and flt tracer accumulations are high , the area is highly malignant as a tumor site and has high proliferative ability .
pet study is a noninvasive examination method , and the use of these tracers in addition to widely used fdg - pet is highly useful for accurate preoperative diagnosis of tumors and identification of tumor area for removal .
we will continue these tracer analyses to elucidate the correlation with pathological diagnosis and radiological diagnosis .
 the use of positron imaging agents such as fdg , met , and flt is expected to lead the way for novel applications toward efficient malignancy grading and treatment of gliomas . in this study , 
 the usefulness of fdg , met and flt - pet images was retrospectively reviewed by comparing their histopathological findings . 
 fdg , met , and flt - pet were performed in 27 patients with who grade iv , 15 patients with who grade iii , and 12 patients with who grade ii during 5.5 years . 
 the resulting pet images were compared by measuring suvs and t / n ratios ( tumor to normal tissue ratios ) . 
 although there were no significant differences in fdg - pet , there were significant differences in the t / n ratios in the met - pet between who grades ii and iv and in the flt - pet between the who grades iii and iv . 
 in glioblastoma patients , the suvs of the areas depicted by mri in the met - pet were different from those suvs in the flt - pet . 
 importantly , the areas with high suvs in both met - pet and flt - pet were also high in ki-67 index and were histologically highly malignant . 
 pet imaging is a noninvasive modality that is useful in determining a tumor area for removal as well as improving preoperative diagnosis for gliomas . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion