ERBB2 (HER2/neu) Oncogene Is Frequently Amplified in Squamous Cell Carcinoma of the Uterine Cervix A. B. Mitra, V. V. V. S. Murty, Mahendra Pratap, et al. Cancer Res 1994;54:637-639. Published online February 1, 1994.
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[CANCERRESEARCH 54,637-639,February1,19941
Advances in Brief
ERBB2
(HER2/neu)
Oncogene Is Frequently
Amplified
in Squamous
Cell
Carcinoma of the Uterine Cervix' A. B. Mitra, V. V. V. S. Murty, Mahendra Pratap, P. Sodhani, and it S. K. Chaganti2 Laboratory
of Cancer Genetics and the Department
of Pathology, Memorial Sloan-Kettering
Cancer Center, New YorlçNew York 10021, [A. B. M., V t V S. M., R. S. K. C./,
and the Institute ofCytology and Preventive Oncology, Indian Council ofMedical Research, NewDelhi India [A. B. M., M. P., P S.J
Abstract
phoresed on 0.8% agarose gels, and transferred onto nylon membranes. The same membranes were repeatedly hybridized, following stripping of probe,
We evaluated a panel of 22 protooncogenes for amplification in 50 primary, untreated squamous cell carcinomas of the uterine cervix. The
with [32PJdCTP-labeled
tumors studied belonged to clinical stages II and III; histologically, the majority of them were moderately to well differentiated. Amplification represented by 5 or more copies was observed for the genes MYCLJ, SEA,
Hybridized
membranes
Betascope
(Betagen)
Posthybridization
washes
were performed
at
sodium dodecyl sulfate for 40 mm.
were exposed
to KOdak XAR film and also read on a
for quantitation
of target gene signals in relation to
control gene signals. The details of 22 protooncogene probes used in this study and their map
CCNDJ, BCLJ, and GLI in one case each (2%); HRAS in 2 cases (4%); and ERBB2 in 7 cases (14%). Amplification of ERBB2 ranged from 5 to 68 copies. In addition, 2 tumors with ERBB2 amplification showed additional restriction fragments suggesting possible mutation or rearrangement of the gene. The high incidence of ERBB2 amplification in cervical cancer suggests that this gene may play an important
probes.
65°Cin 0.1% standard saline-citrate/0.5%
position are listed in Table 1. These probes were obtained either from the American 1)tpe Culture Collection or from the following investigators:
MYCLJ (F. Alt); MYCN (M. Schwab); MDRJ (I. Roninson); MYC (W. Hay ward); HRAS (E. H. Chang); SEA (P. N. Goodfellow); INT2 (0. Peters); CCND1 (A. Arnold); BCLJ (C. M. Croce); KRAS2 (R. A. Weinberg); WNT1 (0. M. Schackleford); GLI (B. Vogelstein); ERBB2 (D. Slamon); and SRC
role in tumorigenesis.
Introduction
(M. Bishop). An MDM2 probe was derived by polymerase chain reaction
Carcinoma of the uterine cervix is the most common cancer which affects women in several parts of the world and causes high mortality. HPV3 16 and 18 have been implicated in the etiology of cervical cancer (1). HPV infection, although apparently necessary, is not con sidered to be sufficient for malignant transformation, suggesting a role for additional host cell genetic factors (1). These genetic factors involved in carcinogenesis of the uterine cervix are still not well understood. Specific chromosomal and molecular abnormalities in cluding i(4p) or i(5p), rearrangements affecting chromosome 1 (2), amplification and overexpression of MYC (3—5),and allelic deletions affecting 3p (6), have been described. Amplification of cellular protooncogenes has been associated with
from a complementary DNA sequence. Two polymorphic probes, D17S4 mapped to 17q and D12S2 mapped to l2p (both obtained from the American Type Culture Collection), and a probe for the immunoglobulin heavy chain
tumor progression
(7), e.g.; MYCN in neuroblastoma
gene joining region gene, .1,, (obtained from J. Ravetch), were used to control DNA loading. Gene copy number was determined as the ratio of target to control
in signals
Materials
cell carcinomas
(8), small cell
of uterine cervix.
Narayan Hospital, New
histologically they were determined to be squamous cell carcinomas. Of the 50
tumors, 2 were poorly differentiated carcinomas, while the remaining were carcinomas.
The ages of the patients
ranged
from 30 to 80 years with a median of 45 years. High molecular weight DNA was isolated from frozen tissues by proteinase
K digestion,phenol-chloroformextractionandethanolprecipitation.Six p@g of DNA were digested with the restriction enzymes EcoRI or HindIII, electro Received 11/5/93; accepted 12/15/93. 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. I Supported
in part requests
3 The
abbreviations
by for
NIH
Grants
reprints
used are: HPV,
CA-05826
should human
and
CA-56125.
be addressed. papillomavirus;
CIN,
cervical
to the same
ratio in placental
were considered
to contain
gene amplification.
were
mapped
to the 11q13
region,
were
coamplified.
ERBB2 also was amplified in the same tumor. Of the 7 tumors with ERBB2 amplification, 6 belonged to clinical stage III and 6 were of well differentiated histology, while one was moderately differentiated. Thmors 13 and 78 showed novel restriction fragments in addition to amplification in EcoRI-digested DNA, while HindIII-digested DNA did not show such additional restriction fragments, suggesting muta tion or rearrangement of ERBB2 gene (Fig. 1).
Delhi, India. Clinically, the tumors studied belonged to stages II or III and
2 To whom
of given genes
SEA, which
to well differentiated
DNA compared
We studied 22 protooncogenes for amplification in DNA isolated
The material studied comprised 50 consecutively ascertained primary un treated tumor biopsies obtained from patients with cancer of uterine cervix
moderately
in tumor
from 50 untreated primary tumors of carcinoma of uterine cervix at clinical stages II or III. Twelve of the 50 (24%) of the tumor DNM showed amplification of 5 or more copies with one or more probes studied. Amplification was observed in one case each (2%) with probes for the gene MYCLJ (16 copies), SEA (13 copies), CCND1 (18 copies), BCL1 (5 copies), and GLI (5 copies); in 2 cases each (2%) with HRAS (5 copies); and in 7 cases each (14%) with ERBB2 (5 to 68 copies) (Table 1; Fig. 1). In one tumor (tumor 59) CCNDJ and
and Methods
seen at the cancer clinic of Lok Nayak Jayaprakash
signals
Results
lung cancer (9), and various other tumor types; and ERBB2 (HER2/ neu) in breast (10), ovarian (11, 12), gastric (13, 14), and bladder carcinomas (15). In the present study we surveyed a panel of 22 protooncogenes and found frequent amplification of the ERBB2 gene in 14% of primary squamous
probe
DNA as described previously (13). Tumors showing 5-fold or more increase
intraepithelial
neoplasia.
Discussion The scope of gene amplification has not been well described in cervical cancer. In order to obtain such data, we evaluated the gene copy number of 22 protooncogenes in 50 tumor specimens obtained from primary, untreated cervical cancer patients. We found that ERBB2 was the most frequently amplified gene in this group of tu mors. The genes MYCLJ, HRAS, SEA, CCNDJ, BCLI, and GLI were rarely amplified. In contrast to previous reports of studies from Eu rope and Mexico (4, 5), we did not find MYC to be amplified in this panel of tumors ascertained in India. The reasons for absence of
637
Downloaded from cancerres.aacrjournals.org on July 15, 2011 Copyright © 1994 American Association for Cancer Research
@
.@__
.@
ERBB2 AMPUFICATION IN CERVICAL CARCINOMA
@
0
i— CSJ (f)
CO
0)w-v-v--w-@-
0
0
C@) 0)
N.
ntoN@r@@N@
CQ 0)
ERBB2 (Hind ID)
@
1, Fig. 1. Amplification
—
—
.•
.HeII
of ERBB2 in squamous
cell carcinoma of uterine cervix. Thmors 7, 13, 22,
a
ERBB2 (EcoRI)
53, and 78 show amplification with high copy num
her. Tumors 13 and 78 show additional EcoRI re striction fragments. Probes D17S4 and D12S2 were
used as controls.
Dl 7S4 @
(EcoRl)
D12S2
(EcoRI) MYC amplification in these tumors are unknown but may be attrib uted to geographic variations of etiology, such as type of HPV infec tion associated with tumor origin. ERBB2 encodes a M@ 185,000 transmembrane protein which is highly homologous to the epidermal growth factor receptor gene which has been mapped to 17q21 (16). Activated ERBB2 has been shown to cause malignant transformation of cells in transfection as says (17). The ligand (gp3O) for the transmembrane tyrosine kinase domain ofERBB2 has been identified which has been shown to inhibit growth of tumor cells which overexpress ERBB2 (18). Although am plification and/or overexpression of ERBB2 has been suggested to be an indicator of poor prognosis in breast, ovarian, and gastric carcino mas (10, 19, 20), its clinical
relevance
and prognostic
significance
has
not been conclusively established. The ERBB2 gene has thus far not been reported to be amplified in squamous cell carcinomas. The frequency of ERBB2 amplification has been reported to vary in dif ferent tumor types and between different studies of the same tumor Table 1 Gene amplification in 50 primary tumors of carcinoma of uterine cervix Locus―
Probe
Map position
MYCLI MYCN RAFJ KIT ROSI
pRll.1 pNB-1 pHEI phckit-l71 phros
3p25 4qll—q22 6q21—q22
EGFR MDRI MET
pE7 S'kc pmet H
7pl2—p13 7q 7q31
MYC HRAS SEA !NT2
JISE p157 p6.2 556
CCNDI BCLJ ETSJ KR4S2 WNTI
No. of cases
lp32
No. of copies
1
16
8q24 llpls.S 11q13 Ilql3
2 1
5 13
pPL-8
llql3
I
18
p94PS pHE 5.4 p5Wll.l p26
Ilql3 11q23.3 12p15.5 12ql3
1
5
ERBB2,
while
the remaining
2 showed
5-fold
amplification.
In our
series, all tumors showing amplification were moderate to well dif ferentiated carcinomas. This is in contrast to the data of Adnane et a!. (23) who reported ERBB2 amplification predominantly in poorly dif ferentiated breast carcinomas. In this series, we investigated only advanced stage tumors. Therefore, it is important to study the ERBB2 amplification and its expression in various stages of CIN in order to understand its role and significance in cervical carcinogenesis and prognosis. The high frequency of ERBB2 amplification observed by us in carcinoma of uterine cervix and overexpression reported in CIN grade 3 (5) suggests that this alteration plays an important role in cervical carcinogenesis. Acknowledgments The authors thank the staff of the cancer clinic, Department of Obstetrics and Gynecology, LNJP Hospital, New Delhi, India, for providing tumor ma
terial. The authors also thank Professor Usha K. Luthra, Kuwait University, for her interest in the study.
MDM2
PCRprobe
12q13
GLI
pKK36P1
l2ql3
1
ERBB2 SRC PDGFB
her2 pHusrcl pSM-1
l7qll.2—ql2 2Oqll.2 22q12.3-q13.1
7
a Details can be found in the report from the Chromosome
type (21). Some of this variation may be due to technical factors as well as the extent of stromal cell contamination in the tumor sample from which DNA was extracted. It is interesting to note that 2 of the 7 tumors with amplified ERBB2 showed additional EcoRI fragments. The sizes of these fragments were dissimilar in the two cases and were amplified severalfold (38 and 68 copies, respectively). They suggest mutations leading to either EcoRI polymorphisms or rearrangements. Previously novel EcoRI restriction fragments for ERBB2 were reported in breast and ovarian carcinomas (12). Attempts to correlate ERBB2 amplification with histological pro gression and prognosis of different tumor systems have yielded in conclusive results (11, 22). Overexpression ofERBB2 was reported in over 60% of the patients with CIN grade 3 and invasive cancers (5). In the present study, S tumors showed >20-fold amplification of
5 5—68
Coordinating
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