Monitoring of response to radiation therapy for human tumor xenografts using 99mTc-HL91 (4,9-diaza-3,3,10,10-tetramethyldodecan-2,11-dione dioxime).

Abstract

Oxygenation status of tumor tissue is an important factor to discriminate it with respect to its radiosensitivity. 99mTc-4,9-diaza-3,3,10,10-tetramethyldodecan-2,11-dione dioxime (99mTc-HL91) is retained in hypoxic tissues, making it possible to use it as hypoxic imaging agent. We evaluated if the accumulation of 99mTc-HL91 in tumors could aid in the prediction of sensitivity of radiation therapy of cancers.
Human tumors (the gastric cancer cell line: MKN45, the epidermoid carcinoma cell line: KB-31, and the lung adenocarcinoma cell line: HLC) were xenografted into the thigh of athymic mice and irradiated with a 4 MV linear accelerator. Tumor growth was measured and 99mTc-HL91 uptakes in tumors were determined by serial imaging, biodistribution, and autoradiography.
99mTc-HL91 uptake (ratio of ROItumor to ROIwhole body) in HLC ranged from 1.1 to 8.0%, and it did not show any response to radiation therapy. Major variations were observed in 99mTc-HL91 accumulation in MKN45 and KB-31; from 0.7 to 4.7%, and from 1.0 to 7.3%, respectively. Some tumors responded to radiotherapy, while others did not. Tumor response was not dependent on the 99mTc-HL91 uptake, tumor size or radiation dose. Comparing 99mTc-HL91 uptake in tumors before (B) and after (A) their radiation, uptake (B) was always smaller than uptake (A) for HLC, and they did not respond to irradiation at all. For MKN45 and KB-31, tumors responded to radiation when their uptake (A) was not higher than uptake (B). In contrast, the tumors continued to grow when their uptake (A) was higher than uptake (B). Sequential 99mTc-HL91 imaging of KB-31 and their autoradiography indicated that tumors whose 99mTc-HL91 uptakes was increased post irradiation were composed of mainly hypoxic cells. On the other hand, many viable areas were observed in tumors when the increase in 99mTc-HL91 uptake was relatively small.
99mTc-HL91 uptake in tumors did not always relate to their sensitivities to radiation therapy. Sequential 99mTc-HL91 imagings post irradiation showed that the increase in 99mTc-HL91 uptake in tumors predicted a poor response to radiation therapy, and that a decrease or no change suggested that radiation therapy would be effective. Monitoring by 99mTc-HL91 imaging is a good tool to predict the radiosentivities of tumors.

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Annals of Nuclear Medicine Vol. 17, No. 2, 131–138, 2003
ORIGINAL ARTICLE
Received December 24, 2002, revision accepted January 27,
2003.
For reprint contact: Takayuki Suzuki, M.D., Department of
Radiology, Keio University School of Medicine, 35 Shinano-
machi, Shinjuku-ku, Tokyo 160–8582, JAPAN.
E-mail: suzuten@ka2.so-net.ne.jp
INTRODUCTION
OXYGENATION STATUS of tumor tissue is an important factor
to determine the radiotherapeutic and chemotherapeutic
effects,1,2 and obtaining information on oxygen condi-
tions of tumors before or during treatment may potentially
predict the subsequent therapeutic efficacy. Tumor cell
structure of circle-wise proliferation especially in the
capillary vessel, which is called tumor code, is seen within
the tumor.3 Two possible mechanisms underlying the
cause of intratumor hypoxia are considered.4 One is that
hypoxia is caused by the limited oxygen diffusion (100–
180 µm) from the capillary vessel to the tissue, which
occurs in two to three layers surrounding the tumor code.
This is called diffusion-limited hypoxia, and hypoxic
Monitoring of response to radiation therapy for human tumor xenografts using
99mTc-HL91 (4,9-diaza-3,3,10,10-tetramethyldodecan-2,11-dione dioxime)
Takayuki SUZUKI, Kayoko NAKAMURA, Takatsugu KAWASE and Atsushi KUBO
Department of Radiology, Keio University School of Medicine
Purpose: Oxygenation status of tumor tissue is an important factor to discriminate it with respect
to its radiosensitivity. 99mTc-4,9-diaza-3,3,10,10-tetramethyldodecan-2,11-dione dioxime (99mTc-
HL91) is retained in hypoxic tissues, making it possible to use it as hypoxic imaging agent. We
evaluated if the accumulation of 99mTc-HL91 in tumors could aid in the prediction of sensitivity
of radiation therapy of cancers. Methods: Human tumors (the gastric cancer cell line: MKN45,
the epidermoid carcinoma cell line: KB-31, and the lung adenocarcinoma cell line: HLC)
were xenografted into the thigh of athymic mice and irradiated with a 4 MV linear accelerator.
Tumor growth was measured and 99mTc-HL91 uptakes in tumors were determined by serial
imaging, biodistribution, and autoradiography. Results: 99mTc-HL91 uptake (ratio of ROItumor to
ROIwhole body) in HLC ranged from 1.1 to 8.0%, and it did not show any response to radiation therapy.
Major variations were observed in 99mTc-HL91 accumulation in MKN45 and KB-31; from 0.7
to 4.7%, and from 1.0 to 7.3%, respectively. Some tumors responded to radiotherapy, while others
did not. Tumor response was not dependent on the 99mTc-HL91 uptake, tumor size or radiation dose.
Comparing 99mTc-HL91 uptake in tumors before (B) and after (A) their radiation, uptake (B) was
always smaller than uptake (A) for HLC, and they did not respond to irradiation at all. For MKN45
and KB-31, tumors responded to radiation when their uptake (A) was not higher than uptake (B).
In contrast, the tumors continued to grow when their uptake (A) was higher than uptake (B).
Sequential 99mTc-HL91 imaging of KB-31 and their autoradiography indicated that tumors whose
99mTc-HL91 uptakes was increased post irradiation were composed of mainly hypoxic cells. On the
other hand, many viable areas were observed in tumors when the increase in 99mTc-HL91 uptake
was relatively small. Conclusion: 99mTc-HL91 uptake in tumors did not always relate to their
sensitivities to radiation therapy. Sequential 99mTc-HL91 imagings post irradiation showed that the
increase in 99mTc-HL91 uptake in tumors predicted a poor response to radiation therapy, and that
a decrease or no change suggested that radiation therapy would be effective. Monitoring by 99mTc-
HL91 imaging is a good tool to predict the radiosentivities of tumors.
Key words: 99mTc-HL91, tumor hypoxia, radiation therapy, prediction, radiosensitivity

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Takayuki Suzuki, Kayoko Nakamura, Takatsugu Kawase and Atsushi Kubo
cells resulting from this mechanism are called chronically
hypoxic cells. The other mechanism is the induction of
acute hypoxic status by transient closure of tumor vessels.
This hypoxic status caused by blood flow interruption is
called perfusion-limited hypoxia, and hypoxic cells re-
sulting from this mechanism are called acutely hypoxic
cells.5 Since Thomlinson et al. have shown that tumor
code 160 µm or less in diameter is not associated with
necrosis, but more than 200 µm-tumor code is always
associated with central necrosis, oxygen concentration of
cancer cells is known to be largely involved in radiosen-
sitivity. The practical proportion of hypoxic cells was
reported to vary widely, in experimentally induced tu-
mors ranging from less than 1% to 80% or more, with a
mean of approximately 15%.6,7 In regard to the relation-
ship between oxygenation condition and radiosensitivity,
biological effect is larger in the presence of oxygen than
in its absence, and oxygen enhancement ratio is 2.5 to 3.0
times higher in the presence than absence of oxygen (i.e.,
oxygen effect), when the radiation dose is high with a low
LET radiation. Such changes in radiosensitivity occur in
oxygen concentrations ranging between 0 and 30 mmHg.
The intermediate sensitivity lying between anoxia and
sufficient oxygen is caused by an oxygen partial pressure
of 3 mmHg.3
It has already been reported that Tc-99m labeled 4,9-
diaza-3,3,10,10-tetramethyldodecan-2,11-dione dioxime
(99mTc-HL91) is a tumor hypoxic marker.8–12 In this
study, we investigated the relationship between 99mTc-
HL91 uptake in tumors and tumor response to radiation in
athymic mice bearing human tumors to determine whether
99mTc-HL91 is useful in predicting radiotherapeutic
efficacy in clinical settings.
MATERIALS AND METHODS
Animal models
Athymic mice implanted with human tumors (the human
gastric tumor cell line MKN45, the human epidermoid
carcinoma cell line KB-31, and the human lung adenocar-
cinoma cell line HLC) were used. Tumor cells of 1 × 107
were subcutaneously inoculated into the thighs of mice,
and when tumor cells grew macroscopically (300–600
mm3), they were used for the experiment. In a KB-31
irradiation study with 20 Gy, smaller sized tumors (100–
200 mm3) were also prepared as experimental material.
99mTc-labeled HL91 for scintigraphy
HL91 was labeled with 99mTc and scintigraphy was con-
ducted as described in our previous paper. Briefly mice
were injected intravenously with 0.1–0.2 ml (37–74 MBq)
of 99mTc-HL91. Imaging was carried out 3 hours later.
Scintigraphy with 99mTc-HL91 was performed just before
irradiation and at the end of the study. In a study of KB-
31 irradiated with 20 Gy, scintigraphy was also performed
on post-irradiation days 4 and 8 in addition to before
irradiation and at the end of study.
Irradiation
Mice were intraperitoneally anesthetized with pentobar-
bital sodium and immobilized with a fixed apparatus, and
the thigh inoculated with tumor cells was irradiated at a
dose rate of 150 cGy/min with a 4 MV linear accelerator
(Mitsubishi ML6M). A bolus material equivalent to wa-
ter, 5 mm in thickness (Floatation Bed Pad: Sakura
IRYOKI) was placed on the leg to be irradiated. Animals
were allocated to six irradiation groups (HLC with 10 Gy,
KB-31 with 10 Gy, MKN45 with 15 Gy, MKN45 with 20
Gy, KB-31 with 20 Gy [large tumors], KB-31 with 20 Gy
[small tumors]) of 2 to 4 each. All values are single
radiation doses. Post-irradiation follow-up was performed
during 28 days in HLC with 10 Gy and KB-31 with 10 Gy,
19 days in MKN45 with 15 Gy, 21 days in MKN45 with
20 Gy, 16 days in KB-31 with 20 Gy (large tumors) and
14 days in KB-31 with 20 Gy (small tumors). The obser-
vation periods were different in each group because the
timing to sacrifice mice was determined by the condition
of the mice. In each irradiation group, 2 to 4 non-irradiated
animals served as a control. Tumor diameters in 3 direc-
tions, length (L), width (W) and height (H), were meas-
ured every 2 or 3 days over a 2- to 4-week period after
irradiation in all animals, including non-irradiated ani-
mals, to evaluate tumor growth. Tumor volumes (mm3)
were calculated on the assumption that the tumors were
hemi ellipsoids where volume equals 4π/3 · L/2 · W/2 · H/2,
that is, approx. 0.5LWH.13,14
Uptake of 99mTc-HL91
Regions of interest (ROI) were defined in the tumor and
the whole body on scintigrams to determine the uptake of
99mTc-HL91. The accumulation ratio of tumor to whole
body (T/WB) was calculated. At end of the study, blood,
muscle, and tumor were obtained to determine the tracer
uptake. The weight and radioactivity of the blood, muscle,
and tumor were measured. The uptake of radioactivity in
Fig. 1 Growth curves of lung adenocarcinoma (HLC).
R1–R3: 10 Gy-irradiated tumors, C: non-irradiated tumor

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Vol. 17, No. 2, 2003
the blood, muscle and tumor (% injected dose/g tissue
[%ID/g]) was obtained, and the tumor-to-blood (T/B)
ratio was also calculated.
Distribution of intratumor 99mTc-HL91
Radioactivity distribution within the tumor was assessed
using autoradiograms and hematoxylin-eosin (HE) stain-
ing of tumor sections, as described in our previous pa-
per.12 Some mice were sacrificed 4 hours after injection of
99mTc-HL91 followed by autoradiography.
RESULTS
Irradiation of the lung adenocarcinoma HLC × 10 Gy
Figure 1 shows growth curves of HLC. R1, R2, and R3 are
tumors exposed to 10 Gy; C is average of non-irradiated
tumors. Tumor growth was monitored over a 4-week
period. All of the irradiated tumors grew as well as non-
irradiated tumor. The lung adenocarcinoma HLC hardly
responded to radiation. The T/B ratio at the end of study
(post-irradiation day 28) was 1.8 for R1, 1.7 for R2, 1.9 for
R3, and 1.7 for C, revealing that the uptake of 99mTc-HL91
in the irradiated group did not differ from that in the non-
irradiated tumors. Tracer uptake in all tumors in the
irradiated group was higher at the end of study than before
irradiation, as evidenced by scintigraphy (T/WB: 1.1%
vs. 3.3% for R1, 1.2% vs. 3.7% for R2, and 1.3% vs. 8.0%
for R3).
Fig. 3 Growth curves of epidermoid carcinoma (KB-31).
R1–R3: 10 Gy-irradiated tumors, C: average of non-irradiated
tumors
Fig. 4 Autoradiograms and HE staining of KB-31 tumor sections (R1, R2 and C).
99mTc-HL91 was strongly accumulated in the border zone.
Fig. 2 Scintigrams of HLC tumors (R3 and C) and autoradio-
grams and HE staining of both tumor sections.

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Takayuki Suzuki, Kayoko Nakamura, Takatsugu Kawase and Atsushi Kubo
Figure 2 shows scintigrams, autoradiograms, and HE
staining of tumor sections in irradiated tumor R3 and non-
irradiated tumor C. As found in scintigraphic findings,
tracer uptake for R3 also visually increased at the end of
study as compared with that before irradiation. Similar
uptake was seen in non-irradiated tumor. Both autoradio-
grams and HE staining of tumor sections indicated low
uptake of 99mTc-HL91 in viable and necrotic areas and an
avid tracer uptake in the border zone suggesting a hypoxic
region.
Irradiation of the epidermoid carcinoma KB-31 × 10 Gy
Growth curves of the epidermoid carcinoma KB-31 are
given in Figure 3. Growth rate was delayed in irradiated
tumors R2 and R3, as compared with non-irradiated
tumors. On the other hand, tumor growth in irradiated
tumor R1 was not inhibited. The T/B ratio of 99mTc-HL91
in the irradiated group (0.7 for R1, 0.4 for R2, and 0.5 for
R3) was lower than that in the non-irradiated tumors (1.7)
at the end of the study (post-irradiation day 28).
Figure 4 shows autoradiograms and HE staining of
tumor sections in irradiated tumors R1 and R2 and non-
irradiated tumor C. Autoradiography revealed a partial
accumulation of 99mTc-HL91 in the lower part of the
tumor in irradiated tumor R1, and generally little accumu-
lation in irradiated tumor R2. The accumulation of 99mTc-
HL91 was relatively noticeable in non-irradiated tumor.
An area where 99mTc-HL91 was not accumulated corre-
sponded to the histologically necrotic area. Low uptake of
99mTc-HL91 in irradiated tumors may have been reflected
by uptake defect in necrotic areas that occurred as a result
of irradiation.
When 99mTc-HL91 uptake was compared in the ROI,
placed on scintigrams, before irradiation and at the end of
study, tumor uptake decreased in tumors whose growth
was inhibited by radiation (T/WB: 7.3% vs. 3.6% for R2
and 1.5% vs. 1.2% for R3). In irradiated tumor R1 in
which tumor-growth inhibition was less than that in the
other two irradiated tumors, T/WB increased from 1.7%
to 2.7%.
The uptake of radioactivity before irradiation was al-
most the same in irradiated tumors R1 (T/WB: 1.7%) and
R3 (T/WB: 1.5%), while it was much higher in irradiated
tumor R2 (T/WB: 7.3%) than in these two tumors. Tumor
response to radiation was markedly poorest in irradiated
tumor R1. There was no correlation between pre-irradia-
tion uptake of 99mTc-HL91 and tumor response to radia-
tion.
Irradiation of the gastric cancer MKN45 × 15 Gy
Figure 5 shows growth curves of MKN45 in 15 Gy-irradi-
ated tumors R1 and R2 and non-irradiated tumors C.
Tumor growth was not inhibited in irradiated tumor R1, as
Fig. 5 Growth curves of gastric cancer (MKN45).
R1–R2: 15 Gy-irradiated tumors, C: average of non-irradiated
tumors
Fig. 6 Scintigrams of MKN45 tumors (R1, R2 and C).

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Vol. 17, No. 2, 2003
well as non-irradiated tumor, but inhibited in irradiated
tumor R2. A decreased uptake of radioactivity for R2 and
a slightly increased uptake for R1 were seen on scintigrams
(Fig. 6). Tracer uptake apparently increased in non-irradi-
ated tumors. The T/B ratio at the end of the study (post-
irradiation day 19) was lower in irradiated tumor R2
(0.68) than non-irradiated tumors (0.95), but did not
greatly differ in irradiated tumor R1 (0.82) from the non-
irradiated tumors. When tumor uptake was compared in
the ROI before irradiation and at the end of the study
(post-irradiation day 19), T/WB decreased from 2.6% to
2.0% for R2 that showed tumor-growth inhibition. In
irradiated tumor R1 whose growth was not inhibited,
however, tumor uptake (T/WB) increased from 3.1% to
4.3%.
Irradiation of the gastric cancer MKN45 × 20 Gy
Figure 7 shows growth curves of the gastric cancer MKN45
for R1, R2, and R3 and C. Tumor growth was inhibited in
irradiated tumors, as compared with the non-irradiated
tumors. Scintigraphy visually revealed that the increase in
tracer uptake was less in irradiated tumor R2 than in non-
irradiated tumors (Fig. 8). Tumor uptake greatly increased
at the end of study (post-irradiation day 21) in the non-
irradiated tumors, as indicated by T/WB (0.8% vs. 3.7%).
In irradiated tumors, on the other hand, tumor uptake only
slightly increased at the end of study, as compared with
before irradiation (T/WB: from 0.7% to 1.1% for R1,
1.6% to 2.3% for R2, and 0.5% to 1.5% for R3). Tumor
uptake increased by 2.9% (3.7–0.8%) at the end of study,
as compared with before irradiation, in the non-irradiated
tumors; however, among the irradiated tumors, tumor R3
exhibited an at most 1.0% (1.5–0.5%) increase in tumor
uptake.
Irradiation of the epidermoid carcinoma KB-31 × 20 Gy
In a KB-31 irradiation study with 20 Gy, tumors were
divided into a small group (100–200 mm3) and large
group (300–600 mm3), because it was expected that
smaller tumors than those used in the above four irradia-
tion studies could be favorably reduced by irradiation and
thus the correlation of tumor reduction with 99mTc-HL91
uptake was made clearer. Furthermore, to predict radio-
therapeutic effects more precisely, tumor uptake was
assessed by performing scintigraphy four times (before
irradiation, 4 and 8 days after irradiation, and at the end of
the study).
Figure 9 shows growth curves in 20 Gy-irradiated large
tumors (similar in size to that in the 10 Gy-irradiation
study). Tumor uptake gradually increased with increasing
tumor size in non-irradiated tumor (T/WB: 1.7%, 2.4%,
2.7% and 3.6% before irradiation, 4, 8 and 16 days after
irradiation respectively). In irradiated tumors R1 (T/WB:
3.7%, 2.2%, 2.8% and 3.3% before irradiation, 4, 8 and 16
days after irradiation respectively) and R2 (T/WB: 2.7%,
2.3%, 3.0% and 4.2% before irradiation, 4, 8 and 16 days
after irradiation respectively) of which growth was not
inhibited, tumor uptake decreased on post-irradiation day
4 but increased on day 8 as compared with before irradia-
tion. Tumor uptake at the end of the study was slightly less
than that before irradiation in irradiated tumor R1. In
Fig. 7 Growth curves of gastric cancer (MKN45).
R1–R3: 20 Gy-irradiated tumors, C: average of non-irradiated
tumors
Fig. 8 Scintigrams of MKN45 tumors (R2 and C).

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Takayuki Suzuki, Kayoko Nakamura, Takatsugu Kawase and Atsushi Kubo
irradiated tumor R2 in which tumor growth was slight,
tumor uptake increased at the end of the study. On the
other hand, in irradiated tumors R3 (T/WB: 3.3%, 1.9%,
1.8% and 1.9% before irradiation, 4, 8 and 16 days after
irradiation respectively) and R4 (T/WB: 3.4%, 2.4%,
2.1% and 1.7% before irradiation, 4, 8 and 16 days after
irradiation respectively) of which growth was inhibited,
tumor uptake plateaued for R3 or gradually decreased for
R4.
Tumor sections were divided into 9 segments in each of
the irradiated tumors. Each segment was microscopically
assessed to identify viable, hypoxic or necrotic status.
Five segments were viable in R1, while R2 consisted of 6
border-zone segments (hypoxic). Five necrotic segments
were observed in R3 and R4. This means that R1 tumor
was mainly composed of viable cells, R2 tumors of mixed
necrotic and viable cells (border-zone), and R3 and R4
tumors of necrotic tumors.
Figure 10 shows growth curves of small tumors ex-
Fig. 9 Growth curves of large tumors (300–600 mm3) of
epidermoid carcinoma (KB-31).
R1–R4: 20 Gy-irradiated tumors, C: average of non-irradiated
tumors
Fig. 10 Growth curves of small tumors (100–200 mm3) of
epidermoid carcinoma (KB-31).
R1–R4: 20 Gy-irradiated tumors, C: average of non-irradiated
tumors
posed to 20 Gy. In a similar manner to large tumors, tumor
uptake increased with increasing tumor size in non-irradi-
ated tumors (T/WB: 1.2%, 2.6%, 2.7% and 3.9% before
irradiation, 4, 8 and 14 days after irradiation respectively).
In irradiated tumors R1 and R2, tumor growth was inhib-
ited a little. In tumor R1 whose growth was comparatively
inhibited (tumor size plateaued), tumor uptake transiently
decreased on post-irradiation day 4 but increased on day
8, with the increase marked at the end of the study (T/WB:
3.1%, 2.5%, 3.4% and 5.3% before irradiation, 4, 8 and 14
days after irradiation respectively). In tumor R2 in which
growth inhibition was minimal (tumor size slightly in-
creased), tumor uptake increased a little (T/WB: 1.4%,
1.4%, 1.5% and 1.9% before irradiation, 4, 8 and 14 days
after irradiation respectively). Tumors R3 and R4 exhib-
ited a marked growth inhibition after irradiation, and
viable tumor cells were detected only in a part of HE
staining of the tumor sections removed at the end of the
study. In these tumors, tumor uptake gradually decreased
(T/WB: 2.3%, 2.0%, 1.6% and 1.0% for R3, 2.2%, 1.4%,
1.2% and 1.0% for R4, before irradiation, 4, 8 and 14 days
after irradiation respectively). Little viable tumor tissue
existed and the majority of the sections consisted muscle
and necrosis in R3 and R4. When tumor sections were
divided into 9 segments for histological assessment, bor-
der-zones of necrotic and viable area were seen in 6 of the
9 segments for R1, viable areas in 6 of the 9 segments for
R2, and necrotic areas in 7 and 9 of the 9 segments for R3
and R4 respectively. That is, R1 tumor was mainly com-
posed of mixed necrotic and viable tissues (border-zone)
and R2 tumors of viable tissues. In tumors R3 and R4, the
majority of the tumor was necrotic, although some parts
remained viable.
DISCUSSION
Assessment of radiosensitivity of cancer before or during
radiation therapy is an urgent issue, and non-invasive
methods should be developed to understand oxygen con-
ditions of tumors clinically. Since misonidazole was
discovered as a hypoxic cell sensitizer about 20 years ago,
several hypoxic cell markers have been developed.
Chapman et al. reviewed the measurement of oxygen
status of tumors and tumor radioresistance by a radionu-
clide imaging technique.15 201Tl has also been investi-
gated as a marker to predict radiosensitivity. A low uptake
of 201Tl has been shown to reflect the presence of non-
viable cells, but it remains undetermined whether tumor
cells are viable or hypoxic when 201Tl is somewhat taken
up by these cells. Fukumoto et al. have also posed a
question as to the usefulness of 201Tl in predicting the
radioresistance of lung cancer.16
Our previous study showed that 99mTc-HL91 was accu-
mulated significantly more in hypoxic areas than in ne-
crotic or viable ones. We have also demonstrated that
uptake of 99mTc-HL91 was less in necrotic areas than in

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Vol. 17, No. 2, 2003
viable ones so that it could distinguish oxidation status in
tumor tissues. The tumor-to-blood ratio of 99mTc-HL91 in
the tumors we studied previously was higher than that of
other 99mTc-labeled hypoxic markers, such as 99mTc-
BMS18132117 and 99mTc-BRU59-21.18 Iodine-123-la-
beled iodoazomycin arabinoside (IAZA) is also reported
to be a hypoxic marker,19,20 but it is unlikely to have any
advantages over 99mTc-HL91.
The following findings were drawn from this experi-
mental study by use of 99mTc-HL91:
1. Accumulation of 99mTc-HL91 in tumors was not
capable of predicting the subsequent tumor response
to radiation, except for tumors such as lung adenocar-
cinoma, HLC, where radioactivity was always avidly
taken up with a poor response to radiation.
2. For tumors where 99mTc-HL91 uptake was increased
post irradiation, growth was not inhibited, meaning
their response to radiation was poor. Autoradiogra-
phy showed the tumor tissue was composed of mainly
hypoxic cells, especially when uptake was highly
increased.
3. Tumor growth was inhibited post radiation, which
was accompanied by decreased tracer uptake in tu-
mors. Autoradiography indicated that the majority of
tumor tissues were necrotic.
4. Serial 99mTc-HL91 imaging showed that a small in-
crease in 99mTc-HL91 uptake suggested a subsequent
poor response to irradiation even though their growth
was delayed transiently. Autoradiography showed
that such tumor tissues still possessed numerous vi-
able cells.
Hypoxic areas in tumor tissue increase in size in paral-
lel with tumor growth, since blood flow fails to provide
sufficient oxygen. When the tumor does not respond to
radiation, it keeps growing and then, its oxygen status
becomes poor. After irradiation of the tumor, its oxygen
status is changed in two ways: to a mixture of hypoxic and
viable cells or one of hypoxic and necrotic ones. It is not
certain what underlies this difference in the response to
irradiation, but our present study indicated these hypo-
thetical ways by serial changes in the 99mTc-HL91 uptake
post radiation. Namely, in the radiosensitive tumors we
studied, their accumulation of 99mTc-HL91 gradually
decreased with reduced tumor size and increase in ne-
crotic areas. On the contrary, in tumors with a relatively
poor response to irradiation, 99mTc-HL91 uptake tran-
siently decreased immediately after radiation, and then
increased, probably because viable cells persisted there.
Furthermore, in tumors with no response to irradiation,
we observed significant increase in 99mTc-HL91 uptake,
probably because tumor tissues were composed mainly of
hypoxic cells.
We speculate about the clinical use of 99mTc-HL91
imaging as follows;
(1) When uptake of 99mTc-HL91 is decreased constantly
post radiation, the tumor would be radiosensitive.
(2) When uptake of 99mTc-HL91 is slightly or tran-
siently decreased, the response to radiation would be
inadequate, and then, repeated irradiation may be of
value.
(3) When uptake of 99mTc-HL91 is significantly in-
creased, the tumor would be resistant to radiation,
and therefore, additional irradiation is unlikely to be
effective.
Further studies are needed to clarify the relation be-
tween radiation effect and hypoxic status under different
conditions.
ACKNOWLEDGMENTS
We thank Amersham International plc. for providing HL91 used
in this study. We also thank Radiological Technologists, Mr.
Ryuichiro Iwasaki, Mr. Isoo Kitagawa and Mr. Hantaro
Yamashita for their support of irradiation and imaging.
This study was supported in part by Grant (11670912,
12877144) from the Ministry of Education.
REFERENCES
1. Gray LH, Conger AD, Ebert M, Hornsey S, Scott OCA.
Concentration of oxygen dissolved in tissues at time of
irradiation as a factor in radiotherapy. Br J Radiol 1953; 26:
638–648.
2. Moulder JE, Rockwell S. Tumor hypoxia: its impact on
cancer therapy. Cancer Metastasis Rev 1987; 5: 313–341.
3. Hall EJ. The oxygen effect and Reoxygenation. In: Radio-
biology for the Radiologist, Philadelphia; Lippincott Will-
iams & Wilkins, 2000: 91–111.
4. Horsman MR. Nicotinamide and other Benzamide analogs
a agents for overcoming hypoxic cell radiation resistance in
tumors. Acta Oncologica 1995; 34: 571–587.
5. Brown JM. Tumor hypoxia, drug resistance, and metastases.
J Natl Cancer Inst 1990; 82: 338–339.
6. Moulder JE, Rockwell S. Hypoxic fractions of solid tumors.
Int J Radiat Oncol Biol Phys 1984; 10: 695–712.
7. Kallman RF. The phenomenon of reoxygenation and its
implications for fractionated radiotherapy. Radiology 1972;
105: 135–142.
8. Cook GJR, Barrington S, Houston S, Maisey MN, Fogelman
I. HL91, a new Tc-99m-labelled agent with potential for
identifying tumour hypoxia: correlation with FDG PET. J
Nucl Med 1996; 37: 87–88.
9. Zhang X, Melo T, Ballinger JR, Rauth AM. Studies of
99mTc-BnAO (HL91): a nonnitroaromatic compound for
hypoxic cell detection. Int J Radiat Oncol Biol Phys 1998;
42: 1–4.
10. Honess DJ, Hill SA, Collingridge DR, Edwards B, Brauers
G, Powell NA, et al. Preclinical evaluation of the novel
hypoxic marker 99mTc-HL91 (Prognox) in murine and xe-
nograft systems in vivo. Int J Radiat Oncol Biol Phys 1998;
42: 731–735.
11. Yutani K, Kusuoka H, Fukuchi K, Tatsumi M, Nishimura T.
Applicability of 99mTc-HL91, a putative hypoxic tracer, to
detection of tumor hypoxia. J Nucl Med 1999; 40: 854–861.
12. Suzuki T, Nakamura K, Kawase T, Kubo A. Biodistribution

Page 8

Annals of Nuclear Medicine
138
Takayuki Suzuki, Kayoko Nakamura, Takatsugu Kawase and Atsushi Kubo
of hypoxic marker, 99mTc-HL91 (4,9-diaza-3,3,10,10-tetra
methyldodecan-2,11-dione dioxime). KAKU IGAKU (Jpn
J Nucl Med) 2001; 38: 333–341.
13. Hopkins HA, Ritenour ER, MacLeod MS, Looney WB. The
effect of varying the time between irradiation and cyclo-
phosphamide on growth response of hepatoma 3924A. Int
J Radiat Oncol Biol Phys 1979; 5: 1455–1459.
14. Looney WB, Hopkins HA, Carter WH Jr. Solid tumor
models for the assessment of different treatment modalities:
XXIII. A new approach to the more effective utilization of
radiotherapy alternated with chemotherapy. Int J Radiat
Oncol Biol Phys 1985; 11: 2105–2117.
15. Chapman JD, Engelhardt EL, Stobbe CC, Schneider RF,
Hanks GE. Measuring hypoxia and predicting tumor ra-
dioresistance with nuclear medicine assays. Radiother Oncol
1998; 46: 229–237.
16. Fukumoto M, Yoshida D, Hayase N, Kurohara A, Akagi N,
Yoshida S. Scintigraphic prediction of resistance to radia-
tion and chemotherapy in patients with lung carcinoma:
technetium 99m-tetrofosmin and thallium-201 dual single
photon emission computed tomography study. Cancer 1999;
86: 1470–1479.
17. Ballinger JR, Kee JW, Rauth AM. In Vitro and In Vivo
Evaluation of a Technetium-99m-Labeled 2-Nitroimidazole
(BMS181321) as a Marker of Tumor Hypoxia. J Nucl Med
1996; 37: 1023–1031.
18. Melo T, Duncan J, Ballinger JR, Rauth AM. BRU59-21, a
Second-Generation 99mTc-Labeled 2-Nitroimidazole for
Imaging Hypoxia in Tumors. J Nucl Med 2000; 41: 169–
176.
19. Mannan RH, Somayaji VV, Lee J, Mercer JR, Chapman JD,
Wiebe LI. Radioiodinated 1-(5-iodo-5-deoxy-beta-D-
arabinofuranosyl)-2-nitroimidazole (iodoazomycin ara-
binoside: IAZA): a novel marker of tissue hypoxia. J Nucl
Med 1991; 32: 1764–1770.
20. Parliament MB, Chapman JD, Urtasun RC, McEwan AJ,
Golberg L, Mercer JR, et al. Non-invasive assessment of
human tumour hypoxia with 123I-iodoazomycin ara-
binoside: preliminary report of a clinical study. Br J Cancer
1992; 65: 90–95.