行政院國家科學委員會專題研究計畫成果報告
計畫名稱-中文: 使 用 鎝 -99m MIBI惡 性 淋 巴 瘤 造 影 結 果 與 P-醣 蛋 白 -多藥 物 抗 藥 性 (Pgp-mdr)及 多藥物抗藥性相關蛋白質(MRP)基因
計畫名稱-英文: The study of technetium-99m-sestamibi scan results in malignant
lymphomas compared with p-glycoprotein expression, multidrug resistance related
protein expression 計畫編號: NSC90-2314-B-039-026-執行期限: 自民國90年8月1日起至民國91年7月31日 主持人: 高嘉鴻 執行機關:中國醫藥學院醫 學 系
一、中文摘要
本研究之目的在利用鎝-99m MIBI 造 影來預估惡性淋巴瘤之化療結果，並與P 醣蛋白（Pgp）及多藥物抗藥性相關蛋白 （MRP）之表現及其他預後因子做比較。 25位惡性淋巴瘤病人於化療前進入本 研究，於靜脈注射鎝-99m MIBI 10分鐘後 進行造影並判讀造影之結果及計算腫瘤背 景之比值（T/B ratio）。所有25個惡性淋 巴 瘤 加 以 切 片 進 行 免 疫 組 織 化 及 分 析 （IHA）以測定Pgp及MRP的表現。並在化療 結束後第1-2年進行化療反應的評估。結果 顯示15位化療反應良好的病人有明顯比較 高的T/B ratio（3.3士0.6對1.2士0.1）。 所有15位化療反應良好的病人有陽性的鎝 -99m MIBI造影結果及陰性的Pgp及MRP表 現。所有10位化療反應不良的病人有陰性 的鎝-99m MIBI造影結果及陽性的Pgp及 MRP表現。但其他的預後因子對於化療反應 並無相關性。因此本研究結果顯示鎝-99m MIBI造影可以呈現Pgp及MRP表現且比較其 它預後因子更能正確預測惡性淋巴瘤化療 的結果。 關 鍵 詞 ： 惡性淋巴瘤、鎝-99m MIBI 造 影、化療、P-醣 蛋 白 表現、 多藥物抗藥 性相關蛋白表現Abstr act
The purpose of this study was to predict
the response of malignant lymphomas (ML)
to chemotherapy by technetium-99m
methoxyisobutylisonitrile (Tc-MIBI) scan
and to compare it with the predictive ability
of P-glycoprotein (Pgp) expression,
multidrug resistance related protein (MRP)
expression and other prognosis factors.
Twenty-five ML patients were enrolled in
this study prior to initiation of chemotherapy.
Images were obtained 10 minutes after
intravenous injection of Tc-MIBI,
interpreted visually and the
tumor-to-background (T/B) ratios calculated.
Immunohistochemical analyses were
performed on sections of the biopsy
specimens to determine Pgp and MRP
expression. Chemotherapy response was
evaluated in the first 1-2 years after
completion of chemotherapy. The mean
T/B ratio of the 15 patients with good
response (3.3 ± 0.6) was significantly higher than that of the 10 patients with poor
response (1.2 ± 0.1). All 15 patients with good chemotherapy response had positive
Tc-MIBI scan results and negative Pgp and
MRP expression. All 10 patients with poor
response had negative Tc-MIBI scan results
and either positive Pgp or MRP expression.
Other prognosis factors showed no
significant difference in the incidence of
good and poor responses. Tc-MIBI scan
results represent Pgp or MRP expression
better than other prognosis factors more
accurately and predict chemotherapy
response in ML patients.
Keywords: Malignant Lymphoma,
Technetium-99m Methoxyisobutylisonitrile,
Chemotherapy Response, P-glycoprotein
Expression, Multidrug Resistance Related
Protein Expression.
Background and Pur pose
Chemotherapy is the primary
therapeutic modality for many maliganat
lymphoma (ML) including all
cases of Hodgkin's disease (HD) [1-3].
Since resistance to chemotherapeutic agents
is a major cause of treatment failure, the
goal of chemotherapy for ML is to avoid
possible resistance and to achieve the
highest response.
The mechanism of tumor uptake of
technetium-99m methoxyisobutylisonitrile
(Tc-MIBI) may involve binding to the
cytosol of the tumor cell [4]. The cationic
charge and lipophilicity of Tc-MIBI,
mitochondrial and plasma membrane
potentials of tumor cells, and cellular
mitochondrial content can all play a
significant role in tumor uptake of this agent
[5], or the uptake may be caused by indirect
phenomena such as increased tumor blood
flow and capillary permeability. Tc-MIBI
scan has been used to successfully predict
the chemotherapy response of ML [6,7].
However, no studies have compared the
relationship between Tc-MIBI scan results
and Pgp or MRP expression in predicting
chemotherapy response of ML. Therefore,
the aim of this study was to compare
Tc-MIBI scan results, immunohistochemical
analyses of Pgp and MRP expression, and
other prognosis factors as predictors of
chemotherapy response in ML patients.
PATIENTS AND METHODS
Patients. Twenty-five patients (13 men, 12
women; age range 25-65 years; mean age:
46.2±12.3 years) with ML (11 with HD and
14 with NHL) were included in the study
and underwent Tc-99m MIBI scans prior to
chemotherapy (Table I). The classification
of ML used followed the Lukes-and-Butler
and updated Kiel systems [8]. After
Tc-MIBI scans, the 11 HD patients received
chemotherapy regimens with nitrogen
mustard (mechlorethamine), vincristine,
procarbazine, and prednisone (MOPP)
alternating with doxorubicin, bleomycin,
vinblastine, and dacarbazine (ABVD); and
the 14 NHL patients received chemotherapy
regimens with cyclophosphamide,
doxorubicin, vincristine, and predinisone
Technetium-99m Methoxyisobutylisonitrile Scan. The imaging procedure began 30
min after oral intake of 500mg perchlorate
prevent abnormal uptake of free Tc-99m
pertechnetate. A commercial MIBI
preparation (max. 5.56Gb (150mCi) in
approximately 1 to 3ml) was obtained from
The Du Pont Merck Pharmaceutical Co.
(Cardiolite, Billerica, MA, USA). The
labeling and quality control procedures were
carried out according to the manufacturer’s
instructions. Labeling efficiencies were all
higher than 95 percent. Each patient was
positioned supine on the imaging table with
the chest strapped to prevent motion.
Because of physiological Tc-MIBI
accumulation in abdominal and pelvic
organs, visualization of ML located in
abdominal and pelvic regions is unreliable.
In this study, images of supradiaphragmatic
ML were obtained 10 minutes after
intravenous injection of 740MBq (20mCi)
Tc-MIBI in the anterior and posterior
projection. The equipment consisted of a
large field-of-view gamma camera fitted
with a low-energy, high-resolution
collimator. A single 20% energy window
was set at 140 keV, and 500K counts were
obtained for each static image.
Tumor-to-background (T/B) ratios were
calculated as the mean counts over the
region of interest (ROI) of the tumor
outlined in the largest lesion ÷ the mean
counts over the ROI of background defined
as the contralateral normal side for the neck
and axilia lesions or normal soft-tissue of
the thorax for mediastinal lesions.
Tc-MIBI uptake in the lesions ≧ axillary
soft-tissue background based on visual
interpretation of at least 2 experienced
nuclear medicine physicians was considered
a positive Tc-MIBI scan results (Figs 1 and
2).
Immunohistochemical Staining.
Formalin-fixed paraffin sections (5-ìm) were
deparaffinized in an oven at 50℃ for 40
concentrations of ethanol-water dilutions.
For MRP immunohistochemical staining,
antigen retrieval was performed by treatment
in citrate buffer in a 700 W microwave oven
for 5 minutes. Endogenous peroxidase was
blocked by 3% hydrogen peroxide for 15
minutes, followed by 5 minutes in phosphate
buffer saline (PBS). The sections were
incubated overnight in a moist chamber at 4
℃ with primary antibody MRP QCRL-1
(10 μ g/ml, Signet Laboratories, Inc.,
Dedham, MA, USA) at 1:100 concentration.
For Pgp immunohistochemical staining,
endogenous peroxidase was blocked by 3%
hydrogen peroxide for 15 minutes.
Antigen retrieval was performed by
treatment with enzyme digestion in 0.1%
trypsin in PBS for 5 minutes at room
temperature and inhibited with 10% skim
milk in PBS for 5 minutes. The sections
were incubated for 2 hours in a moist
chamber at 37 ℃ with primary antibody
JSB-1 (50 μ g/ml, Boehringer Mannheim
Biochemica, Germany) at 1:50 concentration.
After three 5 minute washes in PBS buffer,
detection of the primary antibody was
performed with a link antibody according to
the manufacturer’s instructions (DAKO
LSAB_ 2 System, Peroxidase, Dako
Corporation, Carpinteria, CA, USA) [9-13].
Pgp and MRP expressions were interpreted
by an experienced pathologist blind to
clinical outcome as follows: negative =
less than 10%, positive = 10% or more
stained tumor cells (Figs 3 and 4).
Chemotherapy Response Evaluation. In
this study, chemotherapy response of each
patient was evaluated for the first 1-2 years
after completion of treatment by clinical and
radiological methods such as plain chest
x-ray, chest computed tomography (CT) or
magnetic resonance imaging (MRI), as well
as head and neck CT or MRI, according to
the following scale: 1. Complete response =
no evidence of disease, 2. Partial response =
at least 50% decrease in the sum of the
products of the maximum perpendicular
evidence of progression in any lesion and no
new lesions, 3. No response = less than 25%
increase in the sum of the products of the
maximum perpendicular diameters of all
measurable lesions, no evidence of
progression in any lesion and no new lesions,
and 4. Progressive disease = at least 25%
increase in the sum of the products of the
maximum perpendicular diameters of all
measurable lesions and/or the appearance of
new lesions. We defined complete and
partial responses as good response, while no
response and progressive disease were
defined as poor response.
Statistical Analyses. T/B ratio was
expressed as mean±standard deviation (SD).
A Mann-Whitney U test was used to
evaluate the difference in T/B ratios between
patients with good versus poor response.
The difference in incidence of good and poor
response was evaluated for eight possible
prognosis factors: positive versus negative
Tc-MIBI scan results, positive versus
negative Pgp expression, positive versus
negative MRP expression, HD versus NHL,
stage I-II versus stage III-IV, age > 40 years
versus ≦40 years, and with versus without
B symptoms (night sweats, fever > 38℃for
3 consecutive days, and unexplained weight
loss of >10% body weight) [2,3]. A
Chi-square test was used to determine if the
frequency of good and poor response was
the same for each pair. If the p value was <
0.05, the difference was considered
significant.
Results
Detailed patient data are shown in
Table I. The mean T/B ratio of the 15
patients with good response (3.3 ± 0.6) was
significantly (p < 0.01) higher than that of
the 10 patients with poor response (1.2 ±
0.1). All 15 (100%) patients with good
response had positive Tc-MIBI scan results
and negative Pgp and MRP expression. All
10 (100%) patients with poor response had
negative Tc-MIBI scan results, among who 6
(60%) patients had positive Pgp expression
expression. Tc-MIBI scan results, Pgp
expression, and MRP expression all showed
significant differences in the rate of good
and poor responses. However, no
significant difference in the incidences of
good and poor responses was found for
lymphoma type, stage, age, or B symptoms
(Table II).
Discussion
Our review of previous literature found
only one paper which reported that 17 ML
children with positive Tc-MIBI scan results
and a higher mean T/B ratio had a better
response to chemotherapy than 7 ML
children with negative Tc-MIBI scan results
and a lower mean T/B ratio [7]. Our
results support their findings. However,
their study did not examine the relation
between other prognosis factors, Pgp or
MRP expression and chemotherapy
response.
The mechanism of chemotherapy
resistance in ML is thought to involve
expression of Pgp and MRP [11-14]. The
retention of Tc-MIBI in tumor cells depends
on Pgp and MRP expression, which function
as ATP-dependent efflux pumps for many
chemotherapy agents [15-18]. Therefore,
in this study we used Tc-MIBI scan to
predict the response of ML to chemotherapy.
We found that positive Tc-MIBI scan results
accurately predicted all good chemotherapy
results, which were also related to negative
Pgp and MRP expression. Moreover,
negative Tc-MIBI scan results accurately
predicted poor chemotherapy results in all
patients with positive Pgp or MRP
expression (Table I).
In our previous studies, only early chest
images performed 10 minutes after
intravenous injection of Tc-MIBI proved to
be accurate enough to predict chemotherapy
response in lung and breast cancer
[17,19,20]. Therefore, in this study, we did
not consider it necessary to do delayed chest
imaging to calculate the tumor washout rate
or retention index of Tc-MIBI to predict
chemotherapy response. mRNA
MRP expression in the tumor cell
membrane, Tc-MIBI tumor uptake is
directly based on the Pgp or MRP
expression in the tumor cell membrane, and
it was impossible to extract mRNA from the
formalin-fixed paraffin sections of biopsy
specimens [21-23]. Therefore, we directly
detected Pgp or MRP expression by
immunostaining to correct with Tc-MIBI
tumor uptake (T/B ratio) in our study.
Based on our findings, we conclude
Tc-MIBI scan results can represent Pgp and
MRP expression for predicting
chemotherapy response in ML patients.
However, further studies including larger
case numbers and for patients with
chemotherapy relapses to repeat Tc-MIBI
scan and recheck Pgp or MRP expression
are necessary to confirm our findings.
計 劃 成 果 自 評 本 研 究 之 成 果 能 正 確 解 釋 鎝 -99m MIBI造影正確預測惡性淋巴瘤化療反應結 果的真正機轉，因此鎝-99m MIBI造影將可 成為決定惡性淋巴瘤化療的結果是否有效 的工具。 References
[1] Wilson WH, Chabner BA. Principles
of chemotherapy for lymphomas. In:
The Lymphomas (ed. by G.P. Canellos,
T.A. Lister & J.L. Sklar), Saunders,
Philadelphia. 1998, p. 235.
[2] Neal AJ, Hoskin PJ. Lymphoma. In:
Clinical Oncology: basic principle and practice (ed. by A.J. Neal & P.J.
Hoskin), Arnold, London. 1997, p.
162.
[3] Barr L, Cowan R, Nicolson M.
Heamatological Malignancies. In:
Oncology. (ed. by L. Barr, R. Cowan
& M. Nicolson), Churchill Livingstone,
New York. 1997, p. 150.
[4] Hassan IM, Sahweil A, Constantinides
C, Mahmoud A, Nair M, Omar YT,
Abdel-Dayem HM. Uptake and
kinetics of Tc-99m hexakis 2-methoxy
isobutyl isonitrile in benign and
Clinical Nuclear Medicine
1989;14:333-340.
[5] Chiu ML, Kronauge JF,
Piwnica-Worms D. Effect of
mitochondrial and plasma membrane
potentials on accumulation of hexakis
(2-methoxyisobutylisonitrile)
technetium (I) in cultured mouse
fibroblast. Journal of Nuclear
Medicine 1990;31:1646-1653.
[6] Shih WJ, Rastogi A, Stipp V, Magoun
S, Coupal J. Functional retention of
Tc-99m MIBI in mediastinal
lymphomas as a predictor of
chemotherapeutic response
demonstrated by consecutive thoracic
SPECT imaging. Clinical Nuclear
Medicine 1998;23:505-508.
[7] Kapucu LO, Akyuz C, Vural G, Oguz
A, Atasever T, Buyukpamukcu M,
Unlu M. Evaluation of therapy
response in children with untreated
malignant lymphomas using
technetium-99m- sestamibi. Journal of
Nuclear Medicine 1997;38:243-247.
[8] Jaffe ES. Histopathology of the
non-Hodgkin’s lymphomas and
Hodgkin’s disease. In: The
Lymphomas (ed. by G.P. Canellos, T.A.
Lister & J.L. Sklar), Saunders,
Philadelphia. 1998, p. 77.
[9] Marie JP. P-glycoprotien in adult
hematologic malignancies.
Hematology oncology clinics of North America 1995;9:239-249.
[10] Kostakoglu L, Guc D, Canpinar H,
Kars A, Alper E, Kiratli P, Hayran M,
Gunduz U, Kansu E. P-glycoprotein
expression by technetium-99m-MIBI
scintigraphy in hematologic
malignancy. Journal of Nuclear
Medicine 1998;39:1191-1197.
[11] Yamaguchi M, Kita K, Miwa H, Nishii
K, Oka K, Ohno T, Shirakawa S,
Fukumoto M. Frequent expression of
P-glycoprotein/MDR1 by nasal T-cell
lymphoma cells. Cancer
1995;76:2351-2356.
[12] Niehans GA, Jaszca W, Brunetto V,
Tsuruo T, Bloomfield CD.
Immunohistochemical identification of
P-glycoprotein in previously untreated,
diffuse large cell in immunoblastic
lymphomas. Cancer Research
1992;52:3768-3775.
[13] Webb M, Brun M, McNiven M,
Le-Couteur D, Craft P. MDR1 and
MRP expression in chronic B-cell
lymphoproliferative disorders. British
Journal of Haematology
1998;102:710-717.
[14] Zhan Z, Sandor VA, Gamelin E, Regis
J, Dickstein B, Wilson W, Fojo AT,
Bates SE. Expression of the multidrug
resistance-associated protein gene in
refractory lymphoma: quantitation by a
validated polymerase chain reaction
assay. Blood 1997;89:3795-3800.
[15] Hendrikse NH, Franssen EJ,
van-der-Graaf WT, Vaalburg W,
de-Vries EG. Visualization of
multidrug resistance in vivo. European
Journal of Nuclear Medicine
1999;26:283-293.
[16] Sun SS, Hsieh JF, Tsai SC, Ho YJ, Lee
JK, Kao CH. Expression of mediated
p-glycoprotein multidrug resistance
related to Tc-99m MIBI
scintimamography results. Cancer
Letter 2000;153:95-100.
[17] Vergote J, Moretti JL, de-Vries EG,
Garnier-Suillerot A. Comparison of
the kinetics of active efflux of
99mTc-MIBI in cells with
P-glycoprotein-mediated and
multidrug-resistance
protein-associated
multidrug-resistance phenotypes.
European Journal of Biochemistry
1998;252:140-146.
[18] Hendrikse NH, Franssen EJ,
van-der-Graaf WT, Meijer C, Piers DA,
Vaalburg W, de-Vries EG.
99mTc-sestamibi is a substrate for
P-glycoprotein and the multidrug
resistance-associated protein. British
Journal of Cancer 1998;77:353-358.
[19] Kao CH, ChangLai SP, Chieng PU,
methoxyisobutylisonitrile chest
imaging of small cell lung carcinoma.
Relationship to patient prognosis and
chemotherapy response - a preliminary
report. Cancer 1998;83:64-68.
[20] Kao CH, Hsieh JF, Tsai SC, Ho YJ,
Lee JK. Quickly predicting
chemotherapy response to
paclitaxel-based therapy in non-small
cell lung cancer by early
technetium-99m
methoxyisobutylisonitrile chest
single-photon-emission computed
tomography. Clinical Cancer Research
2000;6:820-824.
[21] Dexter DW, Reddy RK, Geles KG,
Bansal S, Myint MA, Rogakto A,
Leighton JC, Goldstein LJ.
Quantitative reverse
transcriptase-polymerase chain
reaction measured expression of
MDR1 and MRP in primary breast
carcinoma. Clinical Cancer Research
1998;4:1533-1542.
[22] Wang CS, LaRue H, Fortin A, Gariepy
G, Tetu B. mdr1 mRNA expression by
RT-PCR in patients with primary
breast cancer submitted to neoadjuvant
therapy. Breast Cancer Research and
Treatment 1997;45:63-74.
[23] Yokogami K, Kawano H, Moriyama T,
Uehara H, Sameshima T, Oku T, Goya
T, Wakisaka S, Nagamachi S,
Jinnouchi S, Tamura S. Application of
SPET using technetium-99m sestamibi
in brain tumours and comparison with
expression of the MDR-1 gene: is it
possible to predict the response to
chemotherapy in patients with gliomas
by means of 99mTc-sestamibi SPET?
European Journal of Nuclear Medicine 1998;25:401-409.
