Distal Nephron Renal Tumors: Microsatellite Allelotype Thomas J. Polascik, Paul Cairns, Jonathan I. Epstein, et al. Cancer Res 1996;56:1892-1895. Published online April 1, 1996.
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[CANCER RESEARCH 56,1892—1895. April15.19961
Distal Nephron Thomas
Renal Tumors:
J. Polascik,
and Mark
Paul
Cairns,
Microsatellite
Jonathan
I. Epstein,
Allelotype'
Laszlo
Fuzesi,
Jae Y. Ro, Fray
F. Marshall,
David
Sidransky,
Schoenbere
James Buchanan Brady Urological institute and Departments of Urology IT. J. P., F. F. M., M. 5.1, Otorhinolaryngology and Head and Neck Cancer Research Laboratory [P. C., D. S.], and Pathology If. i. E.]. Johns Hopkins University Medical institutions, Baltimore, Maryland 21287-6101: Department of Pathology, The University of Aachen, Aachen, Germany (L F.]; and Department of Pathology, M. D. Anderson Cancer Center, Houston, Texas 77030 Ii. N. R.]
arms 3p, 5q, l7p, and 17q (3). Monosomy of chromosomes 1, 6, 14, 15, and 22 has been observed in CDCs with cytogenetics, and mic rosatellite studies on a limited number of CDCs have demonstrated frequent LOH of chromosomal arms 8p and I 3q (4, 6). To further investigate the molecular events that contribute to the development of distal nephron tumors, we performed a detailed microsatellite allelo type of 18 CDCs and 13 renal oncocytomas using highly informative markers for all autosomal arms.
ABSTRACT Tumors of varying malignant potential arise from the complex epithe hal lining of the nephron. Although the molecular characteristics of renal clear cell carcinomas, which arise from the proximal tubule, have been studied, little is known about tumors that develop from other parts of the renal tubular system. To elucidate common molecular lesions that may contribute to the development or progression of nonproximal tubule renal tumors,
we performed
a detailed
microsateffite
allelotype
of lesions
thought to arise from the renal collecting duct. Eighteen collecting duct carcinomas (CDCs) and 13 renal oncocytomas were studied using highly informative microsateffite markers on all autosomal arms. Loss of het erozygosity (LOH) was identified on multiple chromosomal arms in CDCs and renal oncocytomas. Microsateffite analysis revealed LOH of lq in 57% ofinformative CDCs. LOH wasalsoobservedon arms 6p (45%), Sp (40%), and 21q (40%). In renal oncocytomas,LOll of lq occurred in approximately 30% of tumors, but ip LOH was observed in 57% of informative
cases analyzed.
High levels of LOH were also observed
on
arm 3p was infrequent
in both tumor
types.
Our results
suggest
AND METHODS
Tissue Samples and DNA Extraction. Tumors were obtained from radical nephrectomy
specimens
and preserved
in formalin.
All
specimens
were re
viewedby oneof us (J. I. E.) to confirm the pathologicaldiagnosisof either CDC or oncocytoma. Normal kidney tissue was used as a source of control DNA. Representative areas of tumor and nonneoplastic tissue were serially sectioned in 0.6-p@mincrements with a cryostat. The first and last sections were
arms 8p, 14q, 19q, and 21q in the oncocytomas Studied. Loss of chrome somal
MATERIALS
stained with H&E, and the location of tumor and normal tissue was delineated
that
with ink. Tumor
the molecular events that contribute to the development of distal nephron tumors are distinct from those associated with the efiologj@of proximal tubule renal cancers.
material,
and normal
and samples
were
tissue
placed
were microdissected
in l.5-ml
vials
from surrounding
containing
xylene.
recovered after proteinase K digestion and phenol-chloroform
DNA
extraction
microdissected material. DNA samples were resuspended in buffered medium
as previouslydescribed(7). PCR Amplification and Microsatellite Analysis. Microsatelliteprimers
INTRODUCTION
used for analysis were obtained from Research Genetics (Huntsville,
Approximately 30,600 new cases of renal cancer will be diagnosed in the United States this year, and nearly 12,000 cancer-related deaths will occur as a consequence of renal tumors (1). The majority of renal tumors arise from the epithelium of the proximal renal tubule (RCC3); however, recent studies suggest that renal tumors are biologically heterogeneous. A variety of tumors putatively arising from the distal nephron have been identified. Renal oncocytomas are relatively be nign tumors characterized by cytoplasmic accumulation of eosino philic mitochondria and are thought to arise from the intercalated cells of the renal collecting duct (2, 3). Chromophobe renal carcinomas have cellular characteristics of oncocytomas, are also thought to have a collecting duct progenitor, but follow a clinical course similar to low-grade RCC (3). CDC is a rare, aggressive distal nephron tumor that commonly presents with metastases (4). CDCs are frequently mistaken
for either RCC or transitional
cell carcinoma
of the renal
pelvis preoperatively. Studies of the molecular characteristics of nonproximal tubular renal tumors are limited. Crotty et a!. (5) have demonstrated that oncocytomas are characterized by loss of chromosomes 1 and Y using cytogenetic techniques. RFLP analysis of chromophobe carcinomas has shown that these tumors show frequent LOH of chromosomal Received 11/20/95; accepted 2/14/96. 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
by
a collaborative
research
grant
with
Oncor,
Inc.,
Gaithersburg,
MD. 2 To
whom
requests
was
of the
for
reprints
should
be
addressed,
at
145
Marburg
Building,
Department of Urology, Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, MD 21287-6101. Phone:(410)955-1039;Fax:(410)955-0833.
3The abbreviations usedare:RCC,renalcellcarcinoma; CDC, collectingductcarci noma; LOH, lossof heterozygosity.
Oncor, Inc. (Gaithersburg,
MD), or were synthesized
in our laboratory
AL), (Table
1). Primerswere end labeledwith [‘y-32P-JATP (DuPont New EnglandNu clear, Boston,
MA) using T4 polynucleotide
kinase
(New
England
Biolabs,
Beverly, MA). PCR amplification was performed for 35 cycles with annealing temperatures
ranging
from 50 to 60°C. PCR products
were analyzed
using
electrophoresisin denaturing7% urea-polyacrylamide-formamide gels fol lowed by autoradiography (8). LOH was scored in informative cases if a significant reduction (>30%)
in the signal of the tumor allele was noted in
comparison to the corresponding control allele in the adjacent lane. RESULTS Although multiple chromosomal arms were lost in both types of tumors, microsatellite analysis of CDCs and oncocytomas revealed significantly different patterns of LOH (Figs. 1 and 2). Although multiple chromosomal arms were lost in both types of tumors, CDCs appearedto lose chromosomal arm lq much more frequently than do oncocytomas. Fifty-seven percent (8/12) ofinformative CDCs showed LOH for microsatellite markers mapping to the 1q23 chromosomal band. In contrast, chromosomal arm ip was lost in only 10% of informative CDCs (1/9). Chromosomal arm 6p was lost in 45% of informative CDCs (5/11), and arms 8p and 21q were also lost in at least 40% of the cases examined. LOH, defined as significant reduc tion in an allele from the tumor lane compared to its corresponding control allele, is shown at multiple loci in Fig. 3. In contrast to CDCs, chromosomal arm lq was lost in only 33% of informative oncocyto mas (3/9) but 57% of these tumors lost arm ip (4/7; Fig. 2). Chro mosomal arms 8p, 14q, 19q, and 2lq were all lost in greater than 40% of informative oncocytomas evaluated in this study. Microsatellite instability suggested by the presence of a band shift or the detection of new alleles in the tumor DNA was not noted in these experiments.
1892
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DISTAL NEPHRONRENAL TUMORS Table 1 LOll in human CDC compared to renal oncocytoma Chromosomal location and microsatellite marker identification numbers for markers used in the study. Frequency of LOH and percentage of loss in informative cases is noted for both CDCs
and oncocytomas.
ChromosomeMarkerCDCOncocytomaLOH/inform.a%LOH/inform.%lpD1S2191/911.14/757.1lqD1S1588/1457.13/933.32pD2S1621/166.31/911.12q
D2S125
ND 1/1211.8
D3S10382/17 16.73qD3S12921/137.72/8254pD4S1546 3pD2S143
8.32/12
2/1216.7
01/12
2/98.3
1/1425
7.11/10
2/1010
1/1141.2
9.14/9
0/744.4
D4S1746/1833.31/137.74q D4S171 D5S4172/16 0/1312.5 22.2SqD5S4211/156.71/911.16pD6S2655/1145.51/10106q 5pD4S427
ND
D6S264 207qD7S4861/119.11/119.18p D7S4812/8 7pD6S287
D8S261 D8S2577/17
09p 8qD8S262
D9S1748 33.3lOpD10S2260/1001/333.3l0qDJOSI8S0/1403/1127.3llpD11S9071/119.11/812.5llqD11S9341/166.30/90l2pD12S62211414.33/1127.3l2qD12S950/1401/911.1l3qD13S2704/1233.32/6 16.71/13 3/97.7 GSN5/15 1/633.3 9qD9S1747
D14S267 0l6pD16S4181/156.73/933.316qSPN0/1202/102017p DISSII75/17 l5qD14S288
4/1329.4
30.85/11
Chemoble 9.118pD18S594/1428.63/1127.3l8qDCC2/1513.33/103019pD19S1773/1323.11/911.119qD19S2461/147.13/742.920pD20S571/1 21.41/13 3/1416.7 D17S5792/12 17qTP53
inform.,
D2ISII D22S22826/15
22qD21S259 number
of
informative
1/117.7
19.
12/102020qD2OSIJO0/801/137.721q
22.2a
0/1145.5
21.44/9
3/1440
2/944.4
results.
60
940 .c II 30 Fig. 1. Frequency of allele loss on each chromosomal
arminCDC.
0
E I-, 20
1011111LLIL1L @50 2
3
4
5
6
dlh. 7
8
9 10 11 12 1314 ChromOsome
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Downloaded from cancerres.aacrjournals.org on July 15, 2011 Copyright © 1996 American Association for Cancer Research
15 1617
18 19 20 2122
DISTAL NEPHRONRENAL TUMORS
UP Dq I
0
-J
UI
Fig. 2. Frequency of allele loss on chromosomal arms in human renal oncocytomas.
0
E F-
1
2
3
4
5
6
7
8
9 10 11 12 13 14 1516
17 18 19 20 2122
Chromosome
Allelic imbalance can potentially derive from amplification of one of the two chromosomal homologues rather than from deletion of a specific homologue. However, the ratio of the residual signal from the deleted allele compared to the signal from the retained allele noted in Fig. 3 suggests that LOH is probably responsible for the observed findings. DISCUSSION The search for specific genetic events that contribute to the devel opment of renal cancers has now spanneda decade.The cloning of the VHL gene on chromosomal arm 3p and the recent demonstration that the VHL protein acts as an inhibitor of transcriptional activity pro vides a functional
example
of how
the identification
of specific
genetic lesions can ultimately help explain the pathophysiology of
AcDc12
NT
common diseases (9). Not all renal carcinomas lose chromosome 3p, and an emerging body of data suggest that renal tumors are biologi cally heterogeneous. Based primarily on cytogenetic studies of a limited number of tumors, the most common genetic lesions of distal nephron neoplasms appear to involve low-frequency alterations of several chromosomes. In this study, a comparatively large number of distal nephron lesions was analyzed. LOH of chromosomal arm 3p was observed in
