Download Chromosomal Amplification Is Associated with

[CANCER RESEARCH 5K. 4260-426.1.
October I.
Advances in Brief
Chromosomal Amplification
Germ Cell Tumors1
Is Associated with Cisplatin Resistance of Human Male
Pulivarthi H. Rao, Jane Houldsworth, Nallasivam Palanisamy, V. V. V. S. Murty, Victor E. Reuter, Robert J. Motzer,
George J. Bosl, and R. S. K. Chaganti2
¡Mhoralory of Cancer Genetics. Sloan-Kellering InstÃ-lalefar Cancer Research ¡P.H. R.. J. H.. N. P., R. S. K. CJ anil Departments of Pathology [V. E. R.I, Mediane ¡R.J. M..
C. J. H.¡.timi Httnuin Genetics ¡K.S. K. C.¡.Memorial Hospital. Memorial Sloan-Kellering Cancer Center. New York. New York 10021. und Department oj Pullioloi>\. College of
Physicians antl Surgeons. Coliitnhia University, New York. New York 10032 ¡V.V. V. S. M.]
through overexpression of cyclin D2, encoded by the CCND2 gene,
mapped to this chromosomal arm (2). Although karyotypic analysis of
GCTs occasionally identified the presence at sites other than 12p, of
changes suggestive of gene amplification such as homogeneously
staining regions and double minute chromosomes (3-5), their associ
ation with the resistance phenotype has not been studied. To investi
gate the possible association of chromosomal amplification with cis
platin resistance, we subjected a panel of 34 GCT DNA samples
derived of 17 tumors from relapse-free patients (sensitive) and 17
chemotherapy-resistant tumors belonging to multiple histologies, to
CGH analysis. Our results identified chromosomal amplification (oth
er than at 12pl 1.2-12) in the resistant tumors at the sites: lq31-32,
2p23-24, 7q21, 7q31. 9q22, 9q32-34, 15q23-24. and 20qll.2-12.
These data demonstrate that chromosomal and. hence, gene amplifi
cation may represent an important pathway in GCT resistance to
cisplatin treatment.
Abstract
Chemotherapy resistance of tumors is an important biological and
clinical problem. Studies Troni many tumor types have indicated that
resistance may he hased on multiple genetic pathways. Human male
(¡ermacell tumors (GCTs) are an especially good model system to study
the genetic hasis of tumor sensitivity and resistance to chemotherapy.
GCTs are exquisitely sensitive to treatment with DNA-damaging drugs
such as cisplatin. rarely exhihit / /'5 <gene mutations, express normal p53
protein, and undergo p53-mcdiatcd apoptosis upon drug treatment. A
small proportion of tumors (20-30% of metastatic lesions) escape the
apoptotic response and result in treatment resistance. We have recently
shown (J. Houldsworth, et al.. Oncogene. 16: 2345-2359. 1998) that in a
suhset of such tumors, resistance is linked to TP53 gene mutations. In a
further search for genetic mechanisms underlying resistance, we subjected
a panel of 17 tumors from relapse-free patients (sensitive) and 17 chemo
therapy-resistant tumors to comparative genomic hybridization analysis
to identify possible amplified regions (implying amplified/overexpressed
genes) associated with resistance. With the exception of 12pl 1.2-12, high
Materials and Methods
level amplification was not detected in any of the sensitive tumors. We
have identified eight amplified regions (lq31-32, 2p23-24, 7q21, 7q31,
9q22. 9q32-34, 15q23-24. and 20ql 1.2-12) in five resistant tumors, which
Patient Samples. Tumor biopsy specimens were obtained during diagnos
tic evaluation from patients seen at the Memorial Sloan-Kettering Cancer
suggests that chromosomal and. hence, gene amplification may comprise
a pathway to drug resistance. Identification of amplified/overexpressed
genes at these sites may elucidate new genetic pathways of chemotherapy
resistance in GCTs and possibly also in other tumors.
Center (New York). A total of 34 tumors from 32 patients comprised the study
group. The sensitive cohort included 17 tumors (7 teratomas. 6 seminomas. 2
embryonal carcinomas. 1 yolk sac tumor, and 1 mixed tumor) resected from
patients that were relapse-free as a result of surgical resection alone, of
chemotherapy alone, or of surgical resection combined with adjuvant chemo
therapy (6). Of the 17 sensitive tumors, 9 were studied at diagnosis before
chemotherapy and 8 were studied after chemotherapy. Although all of the
sensitive patients presented with primary teslicular tumors. 6 of the specimens
studied were testicular lesions and 11 were retroperitoneal masses. Tumors
resected from patients who either did not respond to cisplutin-based chemo
Introduction
Human mule GCTs1 arise in spermatogenous cells, display an array
of histopathological differentiation patterns that resemble stages of
embryonal development, and exhibit an exquisite sensitivity to a
variety of chemolherapeutic agents, notably cisplatin and related
comp< ids ( 1). Presently, more than 90% of all newly diagnosed
patiei
-nd 70-80'7r of patients with metastatic disease are cured ( 1).
therapy or responded and then relapsed comprised the resistant cohort (6). Of
the 17 resistant cases (5 teratomas. 4 malignant transformations. 3 yolk sac
tumors. 3 seminomas. and 2 choriocarcinomas) used in this study. 4 were
studied pretreatment, and 13 were studied posttreatment. Five of the tumor
specimens studied were primary mediastinal lesions, and four were primary
testicular lesions. The remaining eight patients presented with primary testic
ular lesions, but the specimens studied were derived from seven retroperitoneal
masses and one inguinal mass.
CGH. Tumor DNA was extracted from fro/en tissue and subjected to CGH
analysis as described, with minor modifications (7). Briefly, tumor DNA and
reference DNA (normal male ftbroblast cell line) were labeled by nick trans
lation with fluorescein-12-dUTP and Texas-red-5-dUTP (NEN-Dupont, Bos
The t
ining are at risk for relapse and death because of resistant
diseast The predominant cisplatin sensitivity, coupled with infre
quent but potentially lethal resistance, make these tumors a useful
model system to investigate chemotherapeutic agent sensitivity and
resistance. Chemotherapy resistance is an important cause of cancer
treatment failure and. hence, a major clinical problem in treating this
disease (1).
Virtually all GCTs exhibit increased copy number of 12p which has
been suggested to play a role in the origin of these tumors possibly
ton. MA), respectively. Approximately 2(X)ng each of tumor and reference
DNAs were coprecipitated with 15 ftg of unlabeled human cot-1 DNA (Life
Technologies. Inc., Gaithersburg. MD) and resuspended in the hybridi/ation
mix before hybridization to human metaphase chromosome spreads prepared
from phytohemagglutinin-stimulated
lymphocytes from normal individuals.
After hybridi/ation for 2-3 days, the slides were washed, and the chromo
somes were counterslained with 4'.6-diamino-2-phenylindole
to identify the
Received 7/15/98: accepted 8/17/98.
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 by N1H Grants and grants from the Byrne Fund (to R. S. K. C.).
2 To whom requests for reprints should be addressed, at Memorial Sloan-Kettering
Cancer Center. 1275 York Avenue. New York. NY 10021.
1The abbreviations used are: OCT. germ cell tumor: CGH, comparative
hybridisation.
genomic
chromosomes.
Digital Image Analysis. Blue, green, and red images were captured with a
cooled-charge coupled device camera attached to a Nikon Microphot-SA
4260
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CHROMOSOMAL
AMPLIFICATION
IN RESISTANT
OCTS
of gains and losses between histológica! groups. Fig. 1 summarizes
the recurring losses, gains, and amplifications noted in tumor speci
mens from relapse-free patients (sensitive) and cisplatin-resistant tu
microscope and processed using the Quantitative Image Processing System
(Quips. Vysis, IL). Chromosomal imbalances were detected on the basis of the
ratio profiles deviating from the balance value of greenrred ratio of 1.0. Ratio
values of 1.20 and 0.80 were set as upper and lower thresholds to define gains
and losses, respectively. These threshold levels were thoroughly tested as
reported previously by us (7). Regions near the centromere of chromosomes 1,
9. 13-16. 21, and 22 were not scored for CGH analysis because of the highly
mors. Recurring gains and losses were noted in both groups. Several
of these regions were common to both groups while others were
unique to each group. Thus, +1, +2p, +7p, +8, +12p, +17q, +20q,
- Ip, -4p, - 1Iq, - 13q, - 18q, and - 19q were noted in both groups.
repeated sequences native to these sites. Chromosomal or subchromosomal
gain or loss was considered recurrent if detected in two or more tumors by
CGH. A region was considered amplified when the fluorescence intensity
values exceeded 2.0 and the FITC showed a strong localized signal. For
assignment of high level amplification to the chromosomal band, the peaks of
the ratio profiles were compared to the corresponding 4'.6-diamino-2-
Changes that were not shared between the two groups comprised +2q.
+6p, + 13q, + 18q, - 6q, -lip, - 18p. and - 19p in sensitive tumors
(Fig. IA) and +3, +6q, +14q, +20p. +21. -4q, and -9 in resistant
tumors (Fig. Iß).High level regional amplification of 12pl 1.2-12
phenylindole
us (7).
was noted in two seminomas, one sensitive (287B) and one resistant
(CG 950110-02). In both cases, this amplification was in addition to
banding of individual chromosomes
as previously described by
an increase in the copy number of the entire 12p (Fig. 2). In contrast,
high level amplification affecting chromosomal regions other than
12p were restricted to only five resistant tumors. These comprised:
lq31-32 (249A), 2p23-24 (231B), 7q21 and 7q31 (203B), 9q22
(240A), 9q32-34 (243A), 15q23-24 (203B, 249A), and 20ql 1.2-12
Results
CGH detected copy number changes in all 34 tumors studied.
Overall, gains of chromosomal regions were more common than
losses (142 versus 82). As expected, all tumors exhibited a gain of
12p. In the entire study group, recurring gains other than 12p, affected
chromosomes 1, 2, 3, 6, 7p, 8. 13q, 14q, 17q, 18q, 20, and 21 whereas
losses affected chromosomes Ip, 4, 6q, 9, 11, 13q, 18, and 19.
Because of the small number of cases, no comparison was attempted
Fig. 1. Ideograms comparing the recurring chro
mosomal gains and losses detected by CGH in 17
sensitive GCTs (A) and 17 resistant GCTs (B). Thin
vertical lines on either side of the chromosome
ideogram indicate only recurrent gains (right) or
losses (left} of a chromosome or a chromosomal
region. Gains/losses were considered recurrent if
observed in two or more tumors. Thick lines indi
cate regions of high copy number (amplification) as
defined in the text.
13
14
(243A) (Fig. 2). The histologies of these five tumors included three
malignant transformations (203B, 23IB, 243A), one teratoma (240A),
and one choriocarcinoma (249A). In the case of tumor 203B, it is
interesting that the adjacent teratomatous lesion (203A) from which
15
16
17
18
19
20
21
22
B
45
13
14
15
67
20
21
22
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CHROMOSOMAL
the malignant transformation (rhabdomyosarcoma)
hibit the same high level amplifications.
AMPLIFICATION
IN RESISTANT
OCTS
arose, did not ex
203B
Discussion
Understanding of the genetic mechanisms that underlie tumor cell
sensitivity and resistance to chemotherapeutic agents is important
from the biological as well as the clinical standpoint. Several path
ways of drug resistance have been identified in tumor cells, e.g.,
231B
amplification and/or overexpression of membrane transporter genes
that decrease the intracellular accumulation of drug, such as MDRl/
PGYÌ(8), MRP1 (9, 10), cMOAT (10), and LRP (9, 10); activation
and/or overexpression of genes that act as agonists to apoptosis
signals, such as BCL2 (11), and BCLXL (11); or inactivation of genes
that regulate cell cycle, such as TP53 (12). GCTs are an excellent
model system to study the resistance phenomenon. They exhibit an
240Â
exquisite sensitivity to cisplatin-based chemotherapy that is thought to
result from the elevated expression of wild-type p53 observed in these
tumors (13, 14). Their cellular response to DNA-damaging insult is
apoptosis by a p53-dependent mechanism (15). In such a system,
although resistance is rare, it provides opportunities to propose and
test specific genetic pathways that may mediate resistance. We have
243A
shown recently (15) that one such pathway in GCTs (predominantly
teratomas) is inactivating missense mutations in the TP53 gene. In
addition, in vitro studies of a resistant cell line with a TP53 missense
mutation in one alÃ-eleshowed that although the non-mutated alÃ-ele
apparently was intact, only the mutated form of the mRNA was
transcribed (15). Although this predicted pathway has been validated
in the present study, other mechanisms of resistance remain to be
249A
identified. To this end, we decided to investigate the possible role of
gene amplification in GCT resistance, a phenomenon that is observed
in a number of tumor systems and is associated with tumor progres
15
sion and resistance (16).
In the present study, we subjected a group of tumor specimens from
relapse-free patients (sensitive) and from patients whose tumor ex
CG 950110-02
hibited clinical resistance to cisplatin-based chemotherapy (resistant)
to CGH analysis with the hypothesis that some of the resistant tumors
may exhibit chromosomal amplification in evidence of possible gene
amplification. Although CGH has been shown to be highly sensitive
12
in identifying amplified DNA sequences in tumors (7), this technique
Fig. 2. Partial CGH karyotypes (left) and corresponding ratio profiles (righi) illustrating
has thus far not been widely applied to identify amplified DNA
high level amplification of chromosomal regions in six resistant GCTs. Hyhridired tumor
regions in drug-resistant tumors. To our surprise, in the relatively
DNA was visualized via F1TC (green), and control DNA was visualized via Texas Red (ml).
The average greenired fluorescent signal ratio along the length of the chromosome is shown.
small sample of GCTs studied here, we found several sites of chro
The blue line in the ratio profile represents the mean of 8-10 chromosomes, and the yellowmosomal amplification other than 12p that were restricted to only the
line represents the SD. The vertical red and green bars on the left and righi of the ideogram
indicate threshold values of 0.80 and 1.20 for loss and gain, respectively.
resistant tumors. These data, therefore, comprise the first evidence of
chromosomal amplification in GCTs associated with resistance.
Among the other sites amplified that were restricted to resistant
tumors, several are of further interest. The ßCi-2-relatedgene, MFL1/
and overexpressed (64%) in breast cancer though its role in tumor
BCL2AÌ,has been mapped to 15q23-24 (17), a region that was seen
response is as yet unknown (20). Candidate genes that have been
amplified in two cases in this study. Interestingly, the MFLI gene
noted to be amplified in other tumors or that have been predicted to
product has been demonstrated to suppress p53-induced apoptosis in play a role in cellular resistance have not been mapped to the remain
a manner similar to that of the antagonists BCL2 and BCLXL (18).
ing four sites found to be amplified in resistant GCTs. Further analysis
This gene, therefore, becomes a good candidate for the gene amplified
of all of the sites needs to be performed to test the possible amplifi
in the two resistant GCTs that may contribute towards their observed
cation of the candidate genes mentioned as well as to identify possible
clinical resistance. The MYCN oncogene mapped to the 2p23-24 site,
novel amplified genes.
The regional amplification at 12pll.2-12 is also of interest. In
noted here to be amplified in one resistant mixed GCT, has been
shown to be frequently expressed in seminomas and embryonal car
creased copy number of 12p, either as one or more copies of i( I2p) or
cinomas, although with no evidence of amplification (19). Amplifi
tandem duplications of 12p in situ or elsewhere in the genome, is a
cation of MYCN is known to predict poor outcome in neuroblastoma
nearly universal feature of GCTs (2). In a small proportion of tumors,
additional amplification of 12pl 1.2-12 has been noted (21, 22). In one
(16). The amplification/overexpression
of the MDRI/PGYI gene,
mapped to 7q21, has long been shown to be a mechanism whereby
study, an yeast artificial chromosome clone has been identified that is
tumor cells attain resistance to chemotherapeutic agents (8). A newly
derived from this region (21 ). However, neither a candidate amplified
gene nor the possible association of this amplification with progresidentified gene (AIBI) mapped to 20ql2 is frequently amplified (10%)
4262
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CHROMOSOMAL
AMPLIFICATION
sion, resistance, or other clinical features of GCTs has been addressed
thus far. Our finding of 12pl 1.2-12 amplification in a sensitive as
well as a resistant tumor suggests that this amplification may not be
associated with resistance.
Although male GCTs exhibit a high sensitivity to cisplatin-based
chemotherapy, studies directed at the understanding of the molecular
mechanisms of invariable resistance of the remaining tumors are
under way. Mutations of TP53 gene have now been identified in a
subset of such clinically resistant tumors (15). Among the group of
tumors studied, TP53 gene status was available for 15 of the 17
resistant cases; four had mutations (203A, 203B, 228A, 349A). We
now have identified a possible second mechanism for resistance: gene
amplification. Five tumors in the panel of 17 exhibited high level
amplification; however, the gene(s) involved and its role in resistance
remains to be confirmed/identified. Between the two studies, only one
of these tumors (203B) showed both chromosomal amplification
(7q21, 7q31, 15q22-24) and TP53 mutation (15). Tumors 203A and
203B represented discrete but contiguous teratoma and transformed
rhabdomyosarcoma in the same patient (23). Although both lesions
had the TP53 gene mutation (15), the 7q21, 7q31, and 15q22-24
amplifications were noted only in the rhabdomyosarcoma, suggesting
that TP53 gene mutation may be proximal to amplification in the
resistance pathway. Further studies aimed at examining other cellular
features associated with resistance to DNA-damaging agents, such as
ratios of BCL2 family members and cellular differentiation, may
reveal additional mechanisms whereby resistance can be achieved.
Such studies will also aid in the understanding of clinical resistance in
other tumor systems.
Acknowledgments
We thank Geralyn Higgins for assistance in clinical data management.
20.
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Chromosomal Amplification Is Associated with Cisplatin
Resistance of Human Male Germ Cell Tumors
Pulivarthi H. Rao, Jane Houldsworth, Nallasivam Palanisamy, et al.
Cancer Res 1998;58:4260-4263.
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