[CANCER RESEARCH 50, 3043-3047, May 15, 1990]
Inverse Correlation between Expression of Multidrug Resistance Gene and N-myc
Oncogene in Human Neuroblastomas1
Akira Nakagawara,2 Kenji Kadomatsu, Shin-ichi Sato, Kimitoshi Kohno, Hiroshi Takano, Kohei Akazawa,
Yoshiaki Nose, and Michihiko Kuwano
Departments of Pediatrie Surgery- [A. jV., K. K.J and Medical Information [K. A., Y. N.J, Faculty of Medicine, Kyushu University 60, Fukuoka 812, and Department of
Biochemistry; Oita Medical School, Oita 879-56 [S. S., K. K., H. T.. M. K.], Japan
ABSTRACT
Genomic amplification of V/mr is an important prognostic indicator
in neuroblastoma. The tumors with amplified N-myc are initially sensitive
to chemotherapy but often acquire resistance to therapy, recur, and
ultimately kill the patients. We measured amplification and expression
of N-myc and expression of mdr-1 in 35 surgically resected neuroblastomas, before acquisition of drug resistance and in 4 recurrent tumors
resistant to chemotherapy. The mdr-1 mKNA expression was found to
be inversely correlated with the N-myc expression. The mdr-1 gene
expression was at a low level in advanced stage and histologically
undifferentiated neuroblastomas, the same group of tumors in which V
myc expression is elevated. A significantly better prognosis was noted in
those patients whose tumors had a high level of mdr-1 expression and a
low level of N-myc expression. The role, if any, of increased expression
of mdr-1 in the acquisition of multidrug resistance in neuroblastoma
remains unclear. However, the aggressive clinical behavior associated
with N-myc amplification and/or expression appears to be linked to
down-regulation of mdr-1 expression.
INTRODUCTION
Genomic amplification and expression of N-wyc are closely
correlated with poor prognosis in patients with neuroblastoma
(1,2). Amplification is frequent in the tumors originating from
adrenal gland, in patients over age 1 year, and in the advanced
stages (3). It also occurs in tumors with a poorly differentiated
histology (4). Our studies revealed that recurrent neuroblastoma
with N-myc amplification no longer responds to chemotherapy,
even though the initial response was good (3). Thus, drug
resistance might be one of the main characteristics related to
N-wyc expression in neuroblastomas with a poor prognosis.
The mdr-1 gene was first isolated by Roninson et al. (5) from
multidrug resistant human KB carcinoma cell line. It is tran
scribed into a 4.5-kilobase mRNA, and the protein product is
the A/r 170,000 membrane glycoprotein (pi70 or P-glycoprotein) expressed in multidrug-resistant cell lines. The location
and structure of the P-glycoprotein are consistent with the
function as an energy-dependent drug efflux pump (6).
Fojo et al. (7) reported that the mdr-1 gene was expressed at
a very high level in the normal adrenal gland and that it was
overexpressed in three surgical specimens from patients with
neuroblastoma, but not in three neuroblastoma cell lines. We
examined the amplification and expression of N-myc and the
expression of mdr-1 in the same tumor samples obtained sur
gically. We found an inverse correlation between expression of
N-myc and mdr-1 and that the mdr-1 mRNA was expressed at
a lower level in neuroblastomas in the advanced stage and the
histologically undifferentiated group. An increase in mdr-1
expression after the acquisition of drug resistance could not be
Received 9/5/89: revised 1/19/90.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' Supported in part by Grant-in-Aid for Cancer Research 63015062 from The
Ministry of Education, Science and Culture of Japan.
¡To whom requests for reprints should be addressed.
confirmed. In contrast, mdr-1 behaved like a marker for cell
differentiation, being expressed in the more well-differentiated
neoplasms and down-regulated in the poorly differentiated tu
mors.
MATERIALS
AND METHODS
Patients. Clinical data such as age, sex, stage, primary site, and
histology are given in Table 1. All these Japanese patients were admitted
to the Kyushu University Hospital or to the University-affiliated Hos
pitals from 1979 to 1988. The clinical stages of the disease were
according to the system of Evans et al. (8).
Histológica!subclassification was according to the Histológica!Clas
sification of Tumors in Infancy and Childhood by the Japanese Path
ological Society (9) which was according to the classification by Evans
et al. (10), basing on the order of the degree of differentiation: 1,
ganglioneuroma; 2a, GNB, well differentiated type; 2b, GNB,3 com
posite type; 2c, GNB, poorly differentiated type; 3a, neuroblastoma,
rosette-fibrillary type; 3b, neuroblastoma, round cell type.
Tumor Specimens. Fresh samples were obtained from the primary
tumor of 35 patients at the initial surgery. Tissues with necrosis,
hemorrhage, or calcification were excluded. All samples were stored at
-80°C until the measurements of amplification of N-myc and expres
sions of N-myc and mdr-1. In four cases, tumor samples were obtained
at recurrence or autopsy (Fig. 1).
Based on the recent therapeutic regimen, preoperative chemotherapy
using one or two courses of CPA (40 mg/kg for 2 days) and/or cisplatinum (CDDP, 20 mg/m2 for 5 days) plus VM-26 (100 mg/m2) was
given for 18 patients with initially inoperable advanced tumors which
were resected more than 3 weeks after the last administration of the
drug. Radiation therapy was not given preoperatively.
Chemotherapeutic Regimen. For patients in stage I, II, and IV-S,
weekly alternate administration of CPA, 300 mg/m2, and vincristine,
1.5 mg/m2, was done for 2 years. For those in stage III and IV,
chemotherapy was performed according to the previously reported
regimen (11). Briefly, a combination of CPA (40 mg/kg for 2 days),
CDDP (20 mg/m2 for 5 days, days 1 to 5) plus VM-26 (100 mg/m2,
day 7) and Adriamycin (60 mg/m2, day 1) plus DTIC (250 mg/m2, days
1 to 5) was given for at least 24 months.
DNA and RNA Analyses. DNAs were prepared from the tissue as
reported previously based on the method by Blin and Stafford (12).
RNAs were isolated from tumor block by the guanidinium/hot phenol
method of Maniai is et al. (13). RNAs were prepared from two multidrug-resistant cell lines, KB-C1 and VJ-300, which were selected from
human epidermoid cancer KB cell line, and also from a drug-sensitive
revertan! cell line (C1-R2) which was isolated after culturing KB-C1
cells in the absence of colchicine for 3 months (14-16). We also used a
drug sensitive human cancer cell line (HC-7-5) which was isolated from
head and neck tumor (17).
Determination of the number of copies of the N-myc gene was as
described (4). Human placenta! DNA was used as a single copy amount.
For RNA analysis, dot blot nylon filters were prepared with RNAs
from tumor blocks by using a BRL blot apparatus. RNAs were crosslinked to the filter by UV irradiation. A "P-labeled DNA probe (0.8kilobase £coRI-///m/III fragment containing the human multidrug re
sistance 1 coding regions from pMDR 105) was prepared by the random
3 The abbreviations used are: GNB, ganglioneuroblastoma: CPA, cyclophosphamide; CDDP, cis-diamminedichloroplatinum(II).
3043
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EXPRESSION
OF mdr-l AND N-myc IN HUMAN NEUROBLASTOMA
Table I Clinical and laboratory data for 35 cases of neuroblastoma
age BMDP 4F (21) on an IBM system 4381 computer.
N-mycCase1234567891011121314151617181920212223242526272829303132333435Age/sex The life table technique based on the Kaplan-Meier procedure was
used to estimate survival, and the generalized Wilcoxon test was used
HistologyO-CO-SO-SO-CO-SO-SO-CO-SO-CO-SO-SO-SO-SO-SO-RO-SO-SO-SO-SO-SO-CO-SO-SO-SO-SO-SO-SO-SO-SO-SO-RO-SO-SO-SO-S3a3a3a3a3a3a2a3a2a
site"
(copies)
mRN¿c3T2<f32Ceccec36C60e37e133e16e1eIf130e139e322414141404140424e1434e34e34e04e24e24e3424e0454e44e44e2454e2434e24e24e24443
mRNA
for an evaluation of significance (Fig. 3).
(mo)4/M4/M7/M2/F3/F7/F38/F57/F72/F6/M8/F13/M18/M21/F22/F22/M47/M96/F96/M4/M5/F5/M18/M27/M30/F30/M32/M37/M68/F72/M96/M132/M4/M4/M34/MStageII111IIIIIIII11HI
I (mo)Alive
(16)Alive
(48)Alive
+34342434343434e2424e34'24e24e24e14°1424e1414e241+'14e04e2414e1434'14'14'04'2414'04Prognosis
(35)Alive
RESULTS
(13)Alive
(60)Alive
Table 1 shows data on 35 patients with a neuroblastic tumor.
(20)Alive
The number of copies of N-myc DNA and the expression of
(58)Alive
(24)Alive
mdr-1 mRNA were measured in all cases and the N-myc expres
(22)Alive
sion was examined in 29 cases. Representative results of North
(52)Alive
ern blot analysis for mdr-1 expression are shown in Fig. 2. mdr(24)Dead
(7)Dead
1 expression was observed in 32 of 35 cases (91%) and N-myc
(12)Dead
expression
in 25 of 29 cases (86%). The amounts of expression
(20)Alive
of N-myc and mdr-1 were inversely correlated (Table 2). A high
(28)Dead
(12)Recurrent
level ofN-myc expression was observed in the N-n/vt-amplified
(19)Dead
tumors. In 18 samples obtained after preoperative chemother
(40)Dead
(30)Alive
apy, the expression of mRNA was not different from tumors
(42)Alive
obtained from previously untreated patients.
(27)Alive
(52)Dead
Neuroblastomas in stages III and IV had an increased expres
0)Dead( 1
sion of N-myc (Table 3). On the contrary, mdr-1 showed a
(7)Dead
decreased expression in the advanced stage group, especially in
(5)Alive
stage IV and IV-S tumors with N-myc amplification. Thus,
(34)Dead
(14)Dead
neuroblastoma with amplified N-myc shows a tendency toward
(19)Dead
an
increased expression of N-myc and a decreased expression
(61)Dead
6)Alive( 1
of mdr-1.
(16)Dead
A similar inverse correlation between expression of N-myc
(6)Alive
and
mdr-1 was observed with regard to histological grade of
(34)Dead
(11)Dead
differentiation of neuroblastoma. The undifferentiated neuro
(7)
blastoma appeared to have a higher degree of N-myc expression
°O-C,originated from mediastinum: O-S. originated from suprarenal region;
and less expression of mdr-1. This inverse relationship was
O-R. originated from retroperitoneum.
* The abbreviations are defined in "Materials and Methods."
most obvious when the tumors from patients under 1 year of
c Preoperative chemotherapy was performed (see "Materials and Methods").
age were excluded (Table 4).
In tumors from patients under 1 year of age and with a
priming method of Feinberg and Vogelstein (18); pMDR 105 was favorable prognosis, both N-myc and mdr-1 were frequently
provided by Dr. M. M. Gottesman (National Cancer Institute, Be- expressed, although the amount of expression of N-myc was
thesda, MD). The ff-actin probe was prepared as described previously
lower and expression of mdr-1 was higher than in tumors from
(16). Hybridization and washing conditions were as described previ
older patients (Table 1). However, in those over 1 year of age,
ously (19). Experiments were repeated 3 times. Quantitative determi
grades of expressions were random.
nations of autoradiograms were confirmed by densitometnc analysis
N-myc amplification is highly specific to the neuroblastoma
(Shimadzu, dual-wave length, flying-spot scanner CS-9000, at 580 nm).
originating from the suprarenal region. Both N-myc and mdrThe grade of expression of N-myc and mdr-1 was normalized by ß1 are expressed frequently in tumors originating from the
actin. The positivity of expression of N-myc and mdr-1 was graded into
suprarenal region but grades are variable. In tumors originating
6 (0+ to 5+) and 4 (0+ to 3+), respectively.
from other regions, N-myc is expressed weakly, and mdr-1 is
Statistics. Goodman and Kruskal's gamma was used to measure the
degree of association for the two-way cross-classification table (20) expressed strongly (Table 1).
The expression of N-myc and mdr-1 were measured in cases
(Tables 2-4). Computations were carried out using the statistical pack
mdr-1
RNA 2
Fig. 1. Dot blot hybridization of mdr-1
mRNA and N-myc mRNA. Lane I, K. A. (case
16). initial surgical sample; Lane 2. K. A.,
recurrent tumor; Lane 3, T. K. (case 27), initial
surgical sample; Lane 4. T. K., recurrent tu
mor; Lane 5, normal human adrenal gland;
Lane 6, drug-sensitive human tumor KB cells;
Lane 7, drug-sensitive human tumor HC-7-5
cells; Lane 8, patient K. O., recurrent tumor;
Lane 9, patient C. M., recurrent tumor at
autopsy.
8 jig
I
RNA 2 jig
I
N-myc
i 99
Lane
1
234567
i
I
8
9
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EXPRESSION
OF mdr-1 AND tì-mycIN HUMAN NEUROBLASTOMA
I
I
I
I
I
I
I
I
34
6
l
t
4
*
29
1
16
3
9
11
12
23
31
*
17
14
25
7
18
32
28
10
20
21
35
22
24
26
19
I
Fig. 2. Dot blot hybridization of mdr-1
mRNA prepared from each case. RNAs from
tumor blocks were suspended in l MTris-HCI,
pH 7.5. and 6 x standard saline-citrate, heated
at 65°Cfor 10 min. and then chilled in ice.
•III
Samples were applied to the BPL dot blot
apparatus. Numbers cases. *, sample from the
patient with other than neuroblastoma.
I
O
I
O
«
I
O
fi
H
CJ
I
hJ
mRNA0+221
+2
1+
2+3+N-myc
4
KD5+2(2)
2(2)
4
1 (D4+2(2)
32+MD" 13+2
' Numbers in parentheses, number of cases with N-myc amplification. The
degree of association was measured by Goodman and Kruskal's gamma (P =
-0.661. t = -4.33, P< 0.01).
Table 3 Stage and expression of N-myc or mdr-1 in neurohlastomas
mRNAStageII
11
N-myc
+1
4+3(3)°
+12
0+2(2)
0
33
I
fM
Table 2 Correlation between expression of N-myc and mdr-1 in neuroblastoma
mdr-1
mRNA0+
8
52(1)
32+135(1)
III
1
IV
2
2(2)
2(2) 8(3) 2
11
IV-S0+2
13+2
13+
1 (1)5+
Ml)mdr-KDmRNA2+16(2)
°Numbers in parentheses, number of cases with N-myc amplification. The
degree of association between stage and N-myc expression: I' = 0.589, r = 4.243.
P < 0.01. The degree of association between stage and mdr-1 expression: I' =
-0.731,1 = -4.999. />< 0.01.
Table 4 Histológica!grade of differentiation and expression of N-myc or mdr-1
in neurohlastomas
ageN-mycfrom patients over 1 year of
mRNAHistology
mRNA
mdr-1
tumor (histologically ganglioneuroblastoma) was resected after
3 courses of effective high dose chemotherapy (CFA, CDDP,
VM-26). However, the patient relapsed and the recurrent tumor
(histologically neuroblastoma) in the spinal cord was obtained
at autopsy. The expression of mdr-1 of the primary tumor was
3+ and in the recurrence was 1+. Recurrent tumor from patient
(K.O.) was obtained at surgery. The recurrent tumor in the
paraaortic lymph nodes showed no expression of mdr-1; how
ever, the tumor was resistant to chemotherapy, and the boy
died of the progressive disease 2 months after the surgery. The
tumor sample obtained at the initial surgery was not available
for examination. However, it is clear that the clinical drug
resistance of this tumor was not related to mdr-1 expression.
As shown previously in other series of patients (22), we found
that N-myc amplification strongly affected the prognosis of our
patients with neuroblastoma (Table 1). N-myc expression also
correlated with the prognosis; the group with lower expression
had a significantly better survival curve, compared to cases of a
high expression (Fig. 3a). On the contrary, in the group with a
low mdr-1 expression, the survival curve was significantly worse
(Fig. 3Ä).
DISCUSSION
We obtained evidence that mdr-1 expression is inversely
correlated with the N-myc expression in surgically resected
2
neuroblastomas sensitive to anticancer agents such as cyclo2b
1
11
(1)°
phosphamide, m-platinum, VM-26, and Adriamycin. mdr-1
2c
2
1(1)
2(1) 1
was strongly expressed in tumors in early stage and with well3a
12
3(2)
2(2) 2(2) 2(2) 6(2) 2(1) 2(1)
3b
1(1)4+
1 (1)3+3
differentiated histology, while N-myc was strongly expressed in
°Numbers in parentheses, number of cases with N-myc amplification. The
advanced stage tumors with poorly differentiated histology. Nhistology abbreviations are defined in "Materials and Methods." The degree of
myc expression adversely affected the prognosis and mdr-I
association between histology and N-myc expression: r = 0.535,1 = 2.215, P <
0.05. The degree of association between histology and mdr-1 expression: F =
expression favorably affected the prognosis. In patients with
-0.744. / = -3.206, P< 0.01.
neuroblastoma with high mdr-1 expression and low N-myc
expression, the survival curve was significantly better. Since
16 (K. A.) and 27 (T. K.) in the primary tumor early in the there was an inverse correlation between expression of N-myc
course when the chemotherapy was effective at inducing a and mdr-1 in neuroblastomas, N-myc expression may result in
response, and after disease recurrence when many of the anti- down-regulation of mdr-1 expression. A down-modulation
cancer drugs were ineffective. In the case of K. A., expression
functioning within the neuroblastoma has been noted between
expression of N-myc and MHC class 1 antigens (23).
of both genes was only slightly increased. However, no change
in expression of N-myc or mdr-1 was evident in the patient T.
The genomic amplification of N-myc closely correlated with
the poor prognosis of neuroblastoma. As Nisen et al. (24)
K. Another study was done on case 5 (C. M.). The primary
3045
3+2a
0+
1+
2+
5+
0+
1+
2+3(1)
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EXPRESSION
100
OF mdr-i AND N-m>-c IN HUMAN NEUROBLASTOMA
We are also looking at increased epidermal growth factor
receptor expression (28) or increased activity of topoisomerase
II (29) or glutathione 5-transferase (30) as possible factors
contributing to an acquisition of multidrug resistance in refrac
tory neuroblastoma.
L-group (n = 9)
80
S
60
ACKNOWLEDGMENTS
40
L
i i
H-group (n = 20)
The authors would like to thank Dr. Dan Longo (National Cancer
Institute, Frederick, MD) for critical comments and reading of the
manuscript and M. Ohara for reading the manuscript.
20
(P = 0.0031)
12345
REFERENCES
Time in years after
initiation
of therapy
100
Time in years after initiation of therapy
Fig. 3. A, effect of N-ntyc expression on the survival curve of neuroblastoma.
L-group, 0+ to 1+ N-myc expression; H-group, 2+ to 5+ N-m>'c expression. B.
effect of mur-l expression on the survival curve of neuroblastoma. L-group, 0+
to 1+ mdr-l expression; H-group, 2+ to 3+ mdr-I expression.
reported, N-myc expression was frequently observed even in
tumors without N-myc amplification. However, the prognosis
was correlated with the grade of the expression. N-myc expres
sion is decreased with differentiation induced by retinoic acid
(25). The clinical samples also showed a decrease in N-myc
expression in the histologically differentiated neuroblastoma
which also showed an increase in the expression of mdr-l.
These data suggest that the product of the mdr-l gene could be
a differentiation antigen in neuroblastoma. Recently, Bates et
al. (26) reported that the expression of mdr-I in human neuro
blastoma cells in culture was related to the degree of differen
tiation.
Several samples, mostly from tumors in stage III or IV were
obtained after one or two courses of the initial preoperative
chemotherapy of cyclophosphamide, m-platinum, or VM-26.
The tumors at that time were sensitive to such drugs and
decreased in size, and surgery was done more than 3 weeks
after the last administration of the drugs. In the field of pediatrie
oncology, primary surgery for advanced neuroblastoma is often
delayed; from the ethical point of view, it is difficult to procure
tissue samples from the patients in advanced stage who have
not been treated with chemotherapy. Recently, Bourhis et al.
(27) have reported that previous chemotherapy may increase
the level of mdr-l expression in human neuroblastoma. In this
study, however, mdr-l expression was still lower as compared
with N-myc expression in the previously treated advanced
neuroblastoma.
We need further clinical studies on the role of mdr-l gene
expression in the multidrug-resistant neuroblastoma. However,
on the basis of the available data, mdr-I does not appear to be
responsible for the clinical drug resistance seen in advanced
stage, N-myc-amplified, poorly differentiated neuroblastomas.
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EXPRESSION OF mdr-l AND N-myc IN HUMAN NEUROBLASTOMA
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Inverse Correlation between Expression of Multidrug
Resistance Gene and N- myc Oncogene in Human
Neuroblastomas
Akira Nakagawara, Kenji Kadomatsu, Shin-ichi Sato, et al.
Cancer Res 1990;50:3043-3047.
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