Neuroradiology (2001) 43: 1023±1030
DOI 10.1007/s002340100629

S. Nagamachi
S. Jinnouchi
K. Nabeshima
R. Nishii
L. Flores II
T. Kodama
K. Kawai
S. Tamura
K. Yokogami
T. Samejima
S. Wakisaka

Received: 14 November 2000
Accepted: 14 April 2001
Published online: 13 July 2001
 Springer-Verlag 2001)

S. Nagamachi ( ) ´ S. Jinnouchi ´
R. Nishii ´ L. Flores II ´ T. Kodama ´
S. Tamura
Department of Radiology,
Miyazaki Medical College,
Miyazaki 889-1692, Japan
E-mail:snagama@post1.miyazaki-med.ac.jp
Phone: +81-9 85-851510ext.22 44
Fax: +81-9 85-85 71 72
K. Nabeshima
Department of Pathology,
Miyazaki Medical College Hospital,
Miyazaki 889-1692, JapanK. Yokogami ´ T. Samejima ´ S. Wakisaka
Department of Neurosurgery,Miyazaki Medical College,
Miyazaki 889-1692, JapanK. KawaiCentral Research Laboratories,
Miyazaki Medical College,
Miyazaki 889-1692, JapanD IA G N O S T I C NE UR OR A DI O LO G Y

The correlation between 99mTc-MIBI uptake
and MIB-1 as a nuclear proliferation
marker in glioma ± a comparative study
with 201Tl

Abstract Technetium-99m methoxy-isobutylisonitrile (MIBI), likethallium-201 (201Tl), is a highly efficient agent for the diagnosis andmonitoring of glioma tumors. Although 201Tl uptake is known to be
partly associated with proliferative
activity, little is known about thecorrelation between MIBI uptake
and proliferation activity in gliomas.
The current study was performed toassess the correlation between MIBI
uptake and proliferative activities ingliomas, estimated by the monoclonal antibody to Ki-67 antigen(MIB-1) staining method. By comparing the results with those of 201Tl,
we determined which tracer would
be suitable for estimating proliferative activities. Twenty-four presurgical glioma patients (six with lowgrade gliomas, five with anaplastic
astrocytomas, and 13 with glioblastomas) were given MIBI and 201Tl
SPECT. Early (10 min after injection) and delayed images (3 h after
injection) were obtained for both
MIBI and 201Tl scintigraphy. SPECT
parameters, early ratio (ER), delayed ratio (DR), and retention in-

Introduction
Proliferative activity is an important prognostic factor inglioma [1, 2, 3, 4, 5]. Proliferation can be assessed by thedetection of Ki-67, which is a nuclear antigen present inall parts of the cell cycle except for the G0 phase. Conventionally, it is diagnosed immunohistopathologicallydex (RI) were obtained in both
radiopharmaceuticals. All patients
underwent subsequent surgical excision, and the specimens were immunostained for MIB-1. The
proliferative activity was measured
as a percentage positive nuclear areafor MIB-1 (MI; MIB-1 index). To
evaluate the relationship between
the proliferative activity and SPECT
parameters, we performed a correlation analysis. MI correlated with
the MIBI uptake ratio (r = 0.75 for
ER, and r = 0.7 for DR). Both DR
and RI of 201Tl also correlated withMI, but weakly (r = 0.6 for DR, andr = 0.59 for RI). There was no significant correlation between the
MIB-1 index and the other parameters. MIBI-uptake parameters demonstrated a stronger positive
correlation with the MIB-1 index
than that of 201Tl. With the use of
MIBI SPECT, we can estimate theproliferative activity of glioma noninvasively.
Keywords 99mTc-MIBI ´ 201Tl ´
Glioma ´ MIB-1 ´ Proliferative
activity

using a monoclonal antibody for the Ki-67 antigen [1,3, 4, 5, 6, 7, 8]. Because monoclonal antibody to Ki-67
antigen (MIB-1) shows higher affinity for Ki-67 thanthe previously generated MoAb [9, 10, 11], it has
achieved widespread use, and MIB-1 proliferation indices are now assessed routinely in many neuropathology
laboratories [1, 3, 4, 5, 6, 7, 8, 9, 10, 11].

1024

Table 1 Tc-99m-MIBI, Tl-201 uptake parameters in comparison with MIB-1 index (LGAS low-grade astrocytoma, ASA anaplastic astrocytoma, GBM glioblastoma multiforme, ER early uptake ratio, DR delayed uptake ratio, RI retention index)
Case

Age (years)/genderHistology

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

14/M
32/M
39/F
50/F
57/M
60/M
32/M
37/M
53/F
55/M
82/F
43/M
46/M
48/F
53/M
55/M
56/M
61/M
68/M
68/M
69/M
69/M
72/M
73/M

LGAS
LGAS
LGAS
LGAS
LGAS
LGAS
ASA
ASA
ASA
ASA
ASA
GBM
GBM
GBM
GBM
GBM
GBM
GBM
GBM
GBM
GBM
GBM
GBM
GBM

201

TI

MIBI
ER

DR

RI

ER

DR

RI

1.1
1.1
1.1
0.9
1.1
1.7
3.4
4.0
12.3
1.8
7.0
6.9
10.8
6.9
2.2
9.8
6.5
5.9
5.0
4.5
2.5
2.5
6.4
12.2

0.9
1.4
1.1
1.2
1.2
1.5
4.4
4.0
13.3
1.9
6.5
7.3
9.9
4.9
1.6
11.4
7.2
4.8
4.2
4.3
2.6
3.4
7.9
16.0

0.8
1.2
1.0
1.3
1.1
0.9
1.3
1.0
1.1
1.0
0.9
1.1
0.9
0.7
0.8
1.2
1.1
0.8
0.8
0.9
1.0
1.3
1.2
1.3

1.2
1.7
1.1
1.0
1.2
1.1
2.7
3.7
2.9
4.9
3.5
3.6
3.4
2.8
1.5
6.5
4.0
2.7
2.9
1.6
1.7
1.8
3.2
4.2

1.0
1.5
1.0
1.1
0.9
1.2
3.0
3.7
4.8
5.1
3.8
5.6
4.2
3.6
1.7
8.8
5.0
4.6
3.0
1.7
3.0
2.0
4.1
5.7

0.8
0.9
0.9
1.1
0.8
1.0
1.1
1.0
1.6
1.0
1.1
1.5
1.2
1.3
1.1
1.3
1.3
1.7
1.0
1.1
1.8
1.1
1.3
1.4

Non-invasive measurement of tumor cell proliferation has been done using pyrimidine nucleus analogues,
such as 11C-thymidine or 131I-iododeoxyuridine, to assess RNA and DNA [12, 13, 14, 15]. However, such
unique radiopharmaceuticals are limited and restrictedto specific institutes.
Alternatively, 201Tl SPECT has been reported to be a
non-invasive tool not only for the detection of a glioma
but also for the prediction of both tumor type and histological grade [16, 17, 18]. Some investigators havereported a correlation between 201Tl uptake and proliferative activity [2, 8]. However, the supposition isstill controversial [12]. In addition, the 201Tl-SPECT image is relatively poor, due to lower energy and smaller
doses.
Technetium-99m methoxy-isobutylisonitrile (MIBI)
is also a tumor-seeking agent used as a substitute for201
Tl for the scintigraphic detection of a variety of malignant neoplasms [19, 20, 21, 22, 23, 24, 25, 26, 27, 28]. Because of the advantage of its higher photon emissions,with clearer images and higher target-to-background ratios, it is now used for detecting brain tumors and for
distinguishing high-grade from low-grade gliomas [22,23, 24, 25, 27]. In addition, MIBI has the capability of diagnosing the presence of P-glycoprotein-mediated anticancer drug resistance [26, 27, 29]. However, there has
been less research regarding the correlation between
MIBI uptake and cell proliferation in gliomas.

MIB-1
(%)
0.3
0.0
0.1
1.8
0.2
0.3
7.3
2.3
30.0
0.9
6.8
29.0
37.0
15.0
15.0
25.0
15.4
15.0
12.1
3.0
10.8
26.8
17.9
17.0

The current study was designed to evaluate the relationship between MIBI uptake and proliferative activity
as determined by the immunohistochemical MIB-1
staining method. By comparing the results with thoseof 201Tl, we determined which tracer would be more appropriate for estimating proliferative activities.

Materials and methods
The study population consisted of 24 patients with glioma (18 men,six women; mean age 59.4  12.9 years, age range 32 to 77 years).
Gliomas were further classified into low-grade astrocytomas(LGASs; grade I and II lesions) and high-grade or malignant gliomas including anaplastic astrocytomas (ASAs; grade III) and glioblastoma multiforme (GBM; grade IV). Taking into account theresolution of our SPECT device, we included in the study only patients with lesions greater than 2 cm maximum diameter on MRI.
All patients entered a study protocol approved by the institutional
review board (Table 1).
Scintigraphic technique
Dual SPECT scans were obtained at both 10 min (early) and 3 h(delayed) after simultaneous intravenous injections of 600 MBq
of MIBI and 111 MBq of 201Tl. Images were obtained by a dual isotope technique as previously reported [20, 21, 27]. Sixty-four projections of 40 s each were acquired by a triple-head gamma
camera system with low-energy, high-resolution, and fan-beam collimators (Picker, Prism 3000). Three energy windows were used for

1025

Fig. 1 Correlation between
MIB-1 index and MIBI uptake
ratioacquisition; they were set at 72 keV with a 15 % window for 201Tl
images, 90 keV with a 10 % window for scatter images, and141 keV with a 15 % window for 99mTc images. These projection
data were processed with a two-dimensional low-pass filter andthen corrected for the contamination scatter. Image reconstruction
was done by filtered back projection with a ramp filter.
Semi-quantitative analysis
We analyzed SPECT images using MR coronal images for anatomical guidance. A region of interest (ROI) was placed on a coronalslice with maximum lesion uptake. A manual ROI was traced
around the lesion, and a round-shaped ROI with a 10-mm diameter was drawn on the opposite side in the appropriate location asa normal tissue. For each image set, early and delayed, an uptake
ratio was calculated as follows:
Averagecountsinthelesion ROI
Averagecountsinthenormal ROI
A retention index was also calculated [21, 27] as follows:
delayed uptake ratio/early uptake ratio
The current study involved simultaneous dual-isotope imaging,and the raw data at the 72-keV window were thus contaminatedby 99mTc Compton scatter. In addition, the raw data at the 141keV window included a gamma-ray count of 201Tl. To eliminate
this scatter contamination, we corrected the raw data on a pixelby-pixel bias [20, 21, 27]. We set three kinds of energy windows,
72 keV, 90 keV, and 141 keV, for data collection. From phantom
studies, the rate of scatter from the 141-keV in the 90-keV window
was 1.06 times that of the 72-keV window. The correction data,
therefore, can be obtained by the formula a = A±aC, where A
stands for the raw counts in the 72-keV window, C indicates theraw counts in the 90-keV window, and a is the scatter correction
coefficient measured to be 1.06 [20, 21, 27]. Similarly, the crosstalk correction coefficient, b, was measured to be 0.22 [20, 21, 27],and corrected counts in the 141-keV window for the 99mTc image,b, were calculated by the formula b = B±ba, where B stands forthe raw counts in the 141-keV window.
Immunohistochemistry
All patients underwent subsequent surgical excision of their tumors. Hematoxylin-eosin-stained sections were used to identify tumor areas for immunohistochemistry.

After deparaffinization and dehydration, the sections were microwaved in a citrate buffer for 10 min. The staining regimen was
performed at room temperature and consisted of exposure to hydrogen peroxide and a blocking antibody to reduce backgroundstaining, followed by incubation with the primary monoclonal antibodies MIB-1 (Immunotech S. A., Marseilles, France), which recognizes an epitope of the Ki-67 antigen. The primary antibody
was visualized with a biotinylated secondary antibody, the avidinbiotinylated enzyme complex, and a chromogen, diaminobenzidine. Hematoxylin was used as a counterstain. Each step was separated by a wash with a citrate buffer. Positive controls consisted ofa tonsil section, while the primary antibody was replaced with abuffer in negative controls.
Using a high-power field ( 400), we counted nuclear stained
cells. The MIB-1 index (MI) was obtained as the percentage of immunopositive cells from the total cells counted in the visualized
fields of a given section. The number of MIB-1-positive cells per
100 tumor cells was designated as the MI and was also calculated
in at least 1,000 cells.
Statistical analysis
To determine the correlation between the MI and SPECT parameters, we calculated Spearman' s rank correlation coefficient. A P
value of < 0.05 was considered to be significant. Statistical analysis
was performed with StatView version 5 (SAS Institute).

Results
All patients with ASAs and GBM showed an intense
uptake of both radiopharmaceuticals. In most of theGBMs, the MI was higher than 10.0. Although the
ASAs showed a variable MIB-1 value, most of themwere lower than 10.0. Conversely, all LGASs showed
faint or no uptake, and all MIs were lower than 0.5 (Table 1). A linear positive correlation was noted between
the ER of the MIBI and MI (r = 0.75, P < 0.01). The
DR of the MIBI also showed significant linear correlation (r = 0.7, P < 0.01) (Fig. 1).
As regards 201Tl, there was a weak positive correlation between the DR (r = 0.6, P < 0.01) and the MI.
However, no significant correlation was noted between
the ER and MIs (Fig. 2). In contrast to the no-significant

1026

Fig. 2 Correlation between
MIB-1 index and 201Tl uptake
ratio

Fig. 3 Correlation between
MIB-1 index and retention index

correlation between the RI and MIs in MIBI, the RI of201
Tl correlated weakly (r = 0.59, P < 0.01) with the MI(Fig. 3).
Case presentation
Case 1 (case number 13) was a 46-year-old man with
GBM. A Gd-enhanced T1-weighted MRI image revealed an irregularly shaped mixed-component tumor in theright temporal lobe. A prominent uptake of both radiopharmaceuticals within the tumor was noted. Uptake indices (ER, DR, and RI) of MIBI were 10.8, 9.9, and 0.9,
respectively. The 201Tl uptake indices were 3.4, 4.2, and1.2, respectively. Immunostaining with monoclonal antibodies MIB-1 demonstrated numerous MIB-1-positive
cells. The MI was 37 % (Fig. 4).
Case 2 (case number 11) was an 82-year-old woman
with ASA. A Gd-enhanced T1-weighted MRI image
showed a uniform ring-enhanced tumor in the rightfrontal lobe. Both the 201Tl and the MIBI demonstrated
intense uptake in accordance with the tumor. The ER,
DR, and RI were 7.0, 6.5, and 0.9, respectively, in the

MIBI, and 3.5, 3.8, and 1.1, respectively, in the 201Tl.
The MI was 6.8 % (Fig. 5).
Case 3 (case number 4) was a 50-year-old man with
LGAS. The post-contrast T1-weighted MRI image
showed a poorly enhanced tumor in the right frontal
lobe. Neither MIBI nor 201Tl uptakes were noted in thetumor. The ER, DR, and RI were 0.9, 1.2, and 1.3, respectively, in the MIBI, and 1.0, 1.1, and 1.1, respectively, in the 201Tl. The specimen sections showed few
positive immunostaining cells for MIB-1. The MI was
1.8 % (Fig. 6).

Discussion
A quantitative assessment of the proliferative potentials
in a glioma provides direct knowledge about its biological behavior and aids in the finding of an accurate prognosis [1, 3]. Although mitotic figures are a conventional
cellular proliferation index, they are variable in number
in lower-grade tumors, and may even be difficult to findin small biopsies of high-grade tumors [3]. Proliferative
activities, therefore, could not be estimated on routine

1027

Fig. 4 a±c Case 13. A 46-yearold man with GBM. a A postcontrast T1-weighted image revealed an irregularly shaped
mixed-component tumor in theright temporal lobe. b BothMIBI and 201Tl accumulated
prominently, coinciding withthe tumor. The 201Tl uptake isnoted mainly in a lateral site
(left upper: early; right upper:
delayed). Conversely, MIBI accumulated dominantly in the
medial site of the tumor (leftlower: early; right lower: delayed). ER, DR, and RI of
MIBI were 10.8, 9.9, and 0,9,respectively. The 201Tl uptake
indices were 3.4, 4.2, and 1.2,
respectively. c Immunostainingof paraffin sections with a monoclonal antibody MIB-1 ( 40)
showed numerous MIB-1-positive cells, and the MIB-1 index
was 37 %abclight-microscopic histopathological assessment [3]. Recently, cell proliferation activity has come to be estimated by immunohistochemical staining methods using themonoclonal antibody Ki-67 [1, 3, 4, 5, 6, 7, 8, 9, 10, 11].
The antigen is present throughout the G1, S, G2, and M
phases in human cells, but it is absent in the G0 phase.
MIB-1, a monoclonal antibody, was recently usedagainst recombinant parts of the Ki-67 antigen, which
can be regarded as a ªtrueº Ki-67 equivalent reactivewith a nuclear antigen in paraffin-embedded materials.
However, the evaluation of such proliferation markers
with immunohistochemical preparations generally involves time-consuming and tedious cell counting by microscopy, which prevents the routine application of
proliferation markers as diagnostic parameters in mostlaboratories. Such backgrounds demand convenientmethods for the estimation of proliferative activity.
Previously, Oriuchi et al. reported a positive correlation between 201Tl uptake and proliferation activity estimated by BUdR [2] and directed the potentiality of 201Tl
as an effective medium for characterizing tumor proliferation [2]. Nonetheless, the current study emphasized that
MIBI uptake indices showed a more significant correlation with the MIs than with those of 201Tl. Although theauthors have no obvious explanation for the weaker correlation with MIB-1 observed with 201Tl, several points
were considered. Among them is the discordance of theuptake mechanism between each tracer except for thepermeability of the blood±brain barrier. In the uptake of

MIBI, strongly negative plasma-membrane potentials
within the cells resulting from increased cellular metabolism contribute to uptake [30, 31, 32]. In addition, P-glycoprotein plays an important role, particularly, ondelayed imaging [22]. Conversely, 201Tl is taken up by
two or more active transport systems such as the Na+-K+
ATPase membrane-pumping system [16, 17, 18]. Another factor is the difference in intratumoral distribution.
Whereas only a small amount of 201Tl accumulates inside
the mitochondria, the majority of MIBI has an intramitochondrial location [30]. Previously, O'Tuama et al. reported that some LGASs could be detected with 201Tl,
but they could not be detected with MIBI [25]. Althoughsuch a noticeable discrepancy was not noted in the current study, certain differences should exist on histologicallevels, which would explain a stronger correlation between the MIBI uptake and the MI.
Another consequential factor is the difference in thereferencing method used to measure the proliferative
activity. BUdR is specifically incorporated into the
DNA synthetic phase (S-phase) of the cell cycle, andthat predicts only the S-phase fraction [12, 13, 14]. In
contrast, the MIB-1 staining method covered not only
the S-phase but also the G1, G2, and M stages that arealso associated with proliferation during a cell's cycleof division. In addition, the derivation of a pyrimidine
nucleoside such as BUdR to a tumor cell is limited by
the presence of a blood±tumor barrier [12]. Such dissimilarities of referencing methods for cell proliferation

1028

Fig. 5 a±c Case 11. An 82-yearold woman with ASA. a A postcontrast T1-weighted image
showed a uniform ring-enhanced tumor in the right frontal lobe. b Both 201Tl and MIBI
demonstrated an intense uptake in accordance with the tumor (left upper: early 201Tl; rightupper: delayed 201Tl; left lower:
early MIBI; right lower: delayed MIBI). The ER, DR, and
RI were 3.5, 3.8, and 1.1, respectively, in the 201Tl and 7.0,
6.5, and 0.9, respectively, in the
MIBI. c Several positive cells
are observed after MIB-1 staining. The MIB-1 index was 6.8 %

a

b

c

could be responsible for the difference between ourdata and those previously reported by other institutes[2]. The correlation coefficient improved in 201Tl on the
delayed images because of the serial change of tracer
distribution probably due to the phenomenon that 201Tl
was retained in mainly active or viable tumor cells. As
the value of the RI also reflects a washout phase, the
RI of 201Tl is also correlated with MIB-1. In contrast,
the distribution of most MIBI remained unchanged on
the delayed image, particularly in GBM. Because ourprevious research demonstrated that GBM contained
few P-glycoproteins (P-gps) [22, 27], an enhanced washout phenomenon did not occur. Therefore, the RI of
MIBI has no meaning in such conditions. In practicaluse, we can estimate the proliferative activity during
the early image of MIBI without consuming a lot oftime, which is one of the advantages of MIBI.
These results lead to an important clinical implication about therapeutic strategy, namely, that intense
and repeated chemotherapy should be recommended
in the treatment of GBM because of its high proliferative activity. However, in choosing anticancer drugs,
drug resistance related to P-gp may not be taken into account. In contrast, it is difficult to choose an anticancer
drug in LGAS. Because of low proliferative activity, intense anticancer chemotherapy may be unnecessary.
Nevertheless, when chemotherapy is performed, theuse of anticancer drugs with multidrug resistance related to P-gp should be avoided.

The other clinical implication addressed in the current study is prognostic value. Although the malignant
biological behavior of neoplasm is determined by manyfactors [33, 34, 35], the proliferative potential is thestrongest prognostic factor for predicting patient survival [1]. We previously reported that intense MIBI uptake
could predict a poor clinical outcome in GBM [27]. The
enhanced proliferative activity may explain the result.
Recently, [18F]-FDG has come to be used widely toidentify the malignancy grade of lesions and to monitor
therapeutic outcome [17, 36], but it does not correlatewith the proliferative rate [37]. Further studies, however, are necessary to assess the clinical relevance. A combined use of [18F]-FDG with MIBI could be a promising
method for the detection of the characteristics of a glioma, including metabolism and proliferation.
An inevitable study limitation is that tissue samplingis usually performed by referencing the findings of lightmicroscopic examinations, which do not provide direct
information about biological behaviors. In addition, viable cells are not always distributed homogeneously intumor tissue [7]. The possibility of cyclical mitotic activity is also present in immunohistochemical analysis.
Such a sampling error and the limitations of the numberof sampling specimens might also affect the result.
The other limitation is the ROI setting. Although we
traced the ROIs under the reference of MRI, they did
not always accord with tumor contour correctly. In particular, a tumor with low tracer uptake, such as LGAS,

1029

Fig. 6 a±c Case 4. A 50-yearold man with LGAS. a A postcontrast T1-weighted image
showed a poorly enhanced tumor in the right frontal lobe.
b Neither MIBI nor 201Tl uptakes were noted in the tumor(left upper: early 201Tl; right upper: delayed 201Tl; left lower:
early MIBI; right lower: delayed MIBI). The ER, DR, and
RI were 1.0, 1.1, and 1.1, respectively, in the 201Tl and 0.9,
1.2, and 1.3, respectively, in the
MIBI. c The specimen sections
showed no positive immunostaining cells for MIB-1. The
MIB-1 index was 1.8 %

b

a

c

is difficult for ROI tracing. To overcome this drawback,combined CT/SPECT tomography has come to be used[38]. However, it is still available at only a limited number of institutes and requires special equipment andsoftware. In the future, such dual-modality devices will
be widely used and will make the study more reliable.
In conclusion, compared with 201Tl, the degree of
MIBI uptake was considered to be more closely relat-

ed to proliferative activity in glioma. Previously, someadvantages of MIBI over 201Tl, such as its imaging
quality [25] and its being a substrate for P-glycoprotein[26, 27], had been reported. The current study added
another meaningful advantage for MIBI, namely, its
being a clinical marker for the proliferative activity inglioma.

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