ICANCERRESEARCH
56.5044-5046.November1, 19961
High-Density Mapping of Chromosomal Arm lq in Renal Collecting Duct
Carcinoma: Region of Minimal Deletion at 1q32.1-32.2
Gabriel Steiner,1 Paul Cairns, Thomas J. Polascik, Fray F. Marshall, Jonathan I. Epstein, David Sidrausky,
and Mark Schoenberg@
James Buchanan Brady Urological Institute 1G. S.. T. J. P., F. F. M.. M. S.] and Departments
IJ. 1. El, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205
ABSTRACT
MATERIALS
Collecting
neoplasm
duct carcinoma
of distal
nephron
(CDC) of the kidney is a rare nanlignnnt
origin.
Previous
studies
of CDC
have
deletion
In 4 additional
tumors.
Our study
further
showed
region of minimal deletion is located at 1q32.1-32.2. Sixty-nine
that the
percent (9
of 13) of the tumors showed loss of heterozygosity in this area. These data
suggest that a gene or group of genes that contribute to the development
of distal nephron tumors may be located within the 1q32.1-32.2 region.
INTRODUCTION
Received 7/1 1/96; accepted 7/I 8/96.
The costsof publicationof this article weredefrayedin part by the paymentof page
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by Deutsche
Thirteen formalin-fixed
and paraffin-embedded
sam
ples of CDCs were obtained as described previously (9). Paraffin blocks were
recut, and standard H&E staining was performed. Confirmation of the diag
nosis of CDC was made by one of us (J. I. E.). Following microdissection,
DNA was extracted from both neoplastic and nonmalignant renal tissue by
24-h proteinase K digestion, phenol-chloroform, extraction and ethanol pre
cipitation (9).
PCR. DNA derived from nonmalignant and neoplastic tissues was ma
lyzed using 20 microsatellite markers (Research Genetics, Huntsville, AL; and
Oncor, Gaithersburg, MD; see Fig. 2) representing selected loci on chromo
somal arm lq. Annealing temperatures, heterozygosity, sequences, length of
PCR products, and loci were obtained from the Genome Data Base and from
the Utah Marker Development Group (10, 11). These microsatellite markers
were chosen to span the length of the entire chromosomal arm at well-spaced
intervals.
Recent studies have demonstrated that malignant kidney neoplasms
are genetically heterogeneous (1, 2). Renal cancers that have been
studied at the molecular level include RCC,3 transitional cell carci
noma, oncocytoma, papillary renal carcinoma, chromophilic and chro
mophobic renal carcinoma, and CDC (2). Renal oncocytoma, chro
mophobe carcinoma, and CDC are thought to arise from the
epithelium of the collecting tubule. Notwithstanding this apparent
common origin, these tumors exhibit exceptionally diverse clinical
behaviors. Renal oncocytomas generally are benign neoplasms with
almost no propensity for metastasis. Chromophobic carcinomas be
have in a manner similar to that of classic RCC, although some
authors have noted a slightly better prognosis among patients with
chromophobic tumors. In contrast, CDCs generally exhibit aggressive
clinical behavior, and many patients with CDC succumb rapidly to the
effects of distant metastases, which are often apparent at the time of
the initial clinical presentation of the disease (1—7).
Few attempts have been made to describe the molecular character
istics of distal nephron tumors in general or of CDCs in particular.
Frequent loss of chromosomal arms 8p and l3q has been reported in
a small number of the CDCs studied (8). In a detailed allelotype of
distal nephron renal cancers, Polascik et a!. (9) demonstrated LOH on
chromosomal arm lq in 57% of the informative CDCs evaluated.
Other authors have noted that the loss of chromosome 1 commonly
occurs in both CDCs and oncocytomas (1, 6).
These observations suggest that genes that contribute to the devel
opment or progression of CDCs reside on chromosomal arm lq. To
test this hypothesis, we performed high-density mapping of the long
arm of chromosome I in 13 CDCs.
I Supported
and Head and Neck Surgery (P. C., D. S.] and Pathology
AND METHODS
Tissue Specimens.
shown
loss ofheterozygosity on chromosomal arm lq in 57% ofthe cases studied.
To better characterize lq loss in CDC, we performed high-density map
ping of the entire long arm of chromosome 1 in 13 CDC tumor samples.
We observed complete deletion of chromosomal arm lq in S samples and
partial
of Otolaryngology
Forschungsgesellschaft
Grant
STE/775-l.
2 To whom requests for reprints should be addressed, at 145 Marburg Building, The
James Buchanan Brady Urological Institute, The Johns Hopkins Hospital, 600 North
Wolfe Street, Baltimore, MD 21287-2101. Phone: (410) 955-1039; Fax: (410) 955-0833.
3 The abbreviations used are: RCC. renal (clear) cell carcinoma; CDC. collecting duct
carcinoma;LOH. lossof heterozygosity.
One primer of each microsatellite marker was end labeled with [‘y-32P]
AlP
(Amersham,
Arlington
Heights,
IL) using T4-polynucleotide
kinase (Life
Technologies, Inc., Gaithersburg, MD). Fifty ng genomic DNA were subjected
to 35 PCR cycles at a denaturing temperature of 95°Cfor 30 s, followed by
varying
annealing
temperatures
ranging
from 54—58°Cfor 1 mm, an extension
step at 70°Cfor 1 mm, and a final extension step at 70°Cfor 5 mm on Hybaid
(United Kingdom) thermocyclers. PCR products were then separated in dena
turing 7% polyacrylamide-urea-formamide gels at voltages between 1400 and
1900 V. The running distances were calculated according to the expected
lengths of the PCR products. Autoradiography was performed with Kodak
X-OMAT scientific imaging film (Eastman Kodak, Rochester, NY) overnight
at —80°C.
LOH was scored in informative cases if a significant reduction
(>30%) in the signal of the tumor allele was noted in comparison with the
corresponding control allele in the adjacent lane.
RESULTS
We tested 13 primary CDCs for LOH on chromosomal arm lq
using PCR-based microsatellite analysis. Representative autoradio
graphs are shown in Fig. I. A summary of the analysis of allelic losses
on the long arm of chromosome 1 at 20 informative markers is shown
in Fig. 2. Four tumors (tumors 4, 11, 16, and 19) did not exhibit any
LOH (data not shown). In five other tumors (tumors 1, 6, 7, 12, and
17), we found LOH throughout the entire long arm of chromosome 1.
LOH was observed in at least one marker on chromosomal arm lq in
9 of I 3 tumors studied. LOH at markers D]5237, D]5389, and
D]S25] was observed in tumor 10, which delineates the centromeric
and telomeric borders of the region of minimal deletion (Fig. 2). The
centromeric border of the region is flanked by two retentions of
heterozygosity at markers D]S]164 and D]5249. The telomeric bor
der is also flanked by two retentions at markers D]5225 and D]S]540.
Furthermore, tumor 8 defines the telomeric border of the region as
demonstrated in Fig. 1, with adjacent LOH at markers D]5389 and
D]525] followed by adjacent retentions at D]5225 and D]S]540.
LOH in the region of minimal deletion is also noted in tumors 2 and
5 (Fig. 2). A shift consistent with a defect in mismatch repair is
evident in tumor 5. Shifts occurred in each tumor that showed cvi
dence of genetic alteration at D1S389.
5044
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CHROMOSOMAL ARM lq IN RENAL COLLEC11NG DUCT CARCINOMA
10
N
DISCUSSION
8
I
N
T
Recent studies have shown that the molecular characteristics of
distal nephron tumors differ from tumors arising in the proximal renal
tubule (i.e., RCC; Refs. 9 and 12). Monosomy of and LOH on
chromosomal arm 3p as well as changes on the long arm of chromo
some 5 are characteristic of RCC (13, 14). Füzesiet aL (6) described
monosomies of chromosomes 1, 6, 14, 15, and 22 in three CDCs. In
addition, one study has described loss of chromosomal arms 3p, 5q,
F7p, and l7q in a case of chromophobic RCC (1). Studies using such
diverse technologies as fluorescence in situ hybridization and com
parative genomic hybridization have demonstrated loss of chromo
some 1 in renal oncocytomas (15, 16).
In the present study, 5 of 13 informative CDCs (39%) exhibited
LOH throughout the entire long arm of chromosome 1. In the previous
study by Polascik et a!. (9), only I of these 5 tumors displayed LOH
on chromosomal arm ip. Therefore, we consider this tumor mono
somic for chromosome 1.
The patterns of LOH observed in tumors 2, 5, 8, and 10 at markers
D]S237, D]S389, and D]525] suggest the location of a region of
minimal deletion. These markers have been mapped to chromosomal
bands 1q32. 1-32.2. At least 25 genes are located in the region of
minimal deletion defined by this study. These include genes that
encode important proteins, such as renin, the complement cascade
enzymes, and proteins associated with mammalian tight junctions and
the binding of the retinoblastoma gene product (17, 18). Interestingly,
to date, no tumor suppressor genes have been identified within this
region.
Cytogenetic analyses have demonstrated that tumors arising from
11
S.
tt.a
DlSll64
D1S389
IL
D1S249
DlS25l
,@
D1S237
D1S225
t•.a
D1S389
DISI54O
Fig. 1. Micmsaiellite analysis in piimaiy CDC. Centromeric (tumor 10) and telomeric
(tumor 8) breakpoints on chromosomal arm lq in CDC. Arrowheads, loss of an allele
consistentwith the LOH. Two retentionsare demonstratedat markersD151164and D1S249,
followed by two markers displaying LOH at D1S237 and D1S389 in tumor 10. In tumor 8,2
LOH again is shown at the latter markers, followed by two retentions at markers D1S251 and
D1S225. This area maps to lq32.l-32.2 (Fig. 2). N, normal; T, tumor.
Tumorl$
11
1
6
12
—
—
—
—
—
—
—
—
—
—
12
—
21.1
21.2
21.3
@
22
@
23
.
.
Fig. 2. Deletion map of chromosomal
@
ann lq in nine CDCs. Only tumors exhibit-
—
U
—
—
—
25
andmappositionsareillustratedon the left.
—
Solid bar on right. area of minimal deletion
at lq32.l-32.2. U, LOH; 0, retention of
@
NJ, not informative;
@n
—
i
shifts.
—
@
—
41
@
@D
—
—
43
44
=
8
2
—
=
=
=
=
=
=
c@
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=
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1=
=
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cu@@
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@UD
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42.2
42.3
=
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32.3
10
—
@j
c@:a
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7
—
@J —
—
SH,
—
@J
24
ing LOH @e
shown. Microsatellite markers
heterozygosity;
17
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5045
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CHROMOSOMAL
ARM Iq IN RENALCOLLECTINGDUCTCARCINOMA
the renal collecting tubule are frequently characterized by loss of
chromosome 1 in contrast to tumors of proximal tubule origin, in
which loss of 3p is the most common molecular finding (19). With the
notable exception of the von Hippel-Lindau gene and Wilms' tumor
genes, the molecular antecedents of renal cancers have yet to be
determined (20). Further study of more CDCs in particular and of
non-RCC renal tumors in general will be required to more completely
delineate the diverse origins of these clinically important carcinomas.
ACKNOWLEDGMENTS
We gratefullyacknowledgeRisa J. Albertafor a carefuleditorialreview of
the manuscript.
8. Schoenberg, M., Cairns, P., Brooks, J. D., Marshall, F. F., Epstein, J. 1.,Isaacs, %V.B.,
and Sidransky, D. Frequent loss of chromosome arms 8p and l3q in collecting duct
carcinoma (CDC) of the kidney. Genes Chromosomes & Cancer. 12: 76—80,1995.
9. Polascik, T. J., Cairns, P., Epstein, J. I., Ftizesi, L., Ro, J. Y., Marshall, F. F.,
Sidransky, D., and Schoenberg, M. Distal nephron renal tumors: microsatellite
allelotype. Cancer Res., 56: 1892—1895,1996.
10. Genome Database, 0DB (TM). The Human Genome Data Base Project. Baltimore:
The Johns Hopkins University. World Wide Web, http://gdbwww. gdb.org/, 1995.
I I. The Utah Markers Development Group. A collection of ordered tetranucleotide repeat
markers from the human genome. Am. J. Hum. Genet., 57: 619—628,1995.
12. Dobin, S. M., Harris, C. P., Reynolds, J. A., Coffield, K. S., Klugo, R. C., Peterson,
R. F., and Speights, V. 0. Cytogenetic abnormalities in renal oncocytic neoplasms.
Genes Chromosomes & Cancer, 4: 25—31,1992.
13. Morita, R., Ishikawa, J., Tsutsumi, M., Hikiji, K., Tsukada, Y., Kamidono, S., Maeda,
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14. Kovács,A., Störkel,S., Thoenes, W., and Kovács,G. Mitochondrial and chromo
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5046
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.
High-Density Mapping of Chromosomal Arm 1q in Renal
Collecting Duct Carcinoma: Region of Minimal Deletion at 1q32.1
−32.2
Gabriel Steiner, Paul Cairns, Thomas J. Polascik, et al.
Cancer Res 1996;56:5044-5046.
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