Measurement of DNA Copy Number at ... - Cancer Research

[CANCER RESEARCH 60, 5405–5409, October 1, 2000]
Advances in Brief
Measurement of DNA Copy Number at Microsatellite Loci Using Quantitative
PCR Analysis
David G. Ginzinger, Tony E. Godfrey, Janice Nigro, Dan H. Moore II, Seiji Suzuki, Maria G. Pallavicini,
Joe W. Gray, and Ronald H. Jensen 1
Department of Laboratory Medicine, University of California San Francisco Comprehensive Cancer Center, University of California, San Francisco, California 94143
Abstract
This report describes the development and validation of quantitative
microsatellite analysis (QuMA) for rapid measurement of relative DNA
sequence copy number. In QuMA, the copy number of a test locus relative
to a pooled reference is assessed using quantitative, real-time PCR amplification
of loci carrying simple sequence repeats. Use of simple sequence
repeats is advantageous because of the large numbers that are mapped
precisely. In addition, all markers are informative because QuMA does
not require that they be polymorphic. The utility of QuMA is demonstrated
in assessment of the extent of deletions of chromosome 2 in
leukemias arising in radiation-sensitive inbred SJL mice and in analysis of
the association of increased copy number of the putative oncogene
ZNF217 with reduced survival duration in ovarian cancer patients.
Introduction
Analysis of allelic imbalance at specific genomic loci is an important
step in the molecular genetic analysis of human cancers because
recurrent regions of imbalance signal the locations of genes that are
involved in disease progression. Often, analysis of allelic imbalance is
accomplished by analysis of polymorphic microsatellite markers [e.g.,
carrying SSRs 2 (1)]. This approach is powerful because SSRs are
dispersed throughout the genomes of most eukaryotes with an average
spacing of 30 kb (2–4). However, assays of allelic imbalance
typically require allelic heterozygosity so that only a subset of the loci
will be informative in any individual (5). This is particularly limiting
in experimental murine systems using inbred mouse strains that have
lost heterozygosity. Analyses of these mice require cross-breeding to
reintroduce heterozygosity. This is both time-consuming and experimentally
undesirable because the cross-breeding may introduce DNA
sequence variations that alter tumor phenotype.
Here we describe a technique called QuMA that overcomes some of
these limitations by allowing analysis of the relative DNA copy
number at microsatellite markers distributed throughout the genomes
of higher eukaryotes. This real-time, quantitative PCR technique is an
alternative to analysis of allelic imbalance because most (but not all)
allelic imbalance also results in a change in relative copy number. We
demonstrate that QuMA is sufficiently sensitive to detect gain or loss
of a single copy of a test locus in mouse and human. We show the
biological utility of QuMA in localization of a putative tumor suppressor
gene associated with radiation-induced murine myeloid leukemia
on chromosome 2 and in establishing an association between
Received 5/5/00; accepted 8/16/00.
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.
1 To whom requests for reprints should be addressed, at University of California San
Francisco Comprehensive Cancer Center, Cancer Genetics and Breast Oncology Program,
2340 Sutter Street, Room S431, San Francisco, CA 94143.
2 The abbreviations used are: SSR, simple sequence repeats; QuMA, quantitative
microsatellite analysis; FAM, 6-carboxy fluorescein; TI, tolerance interval; CGH, comparative
genomic hybridization; FISH, fluorescence in situ hybridization; BAC, bacterial
artificial chromosome; Ct, threshold cycle; MDA 361, MDA-MB-361.
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increased copy number of the putative oncogene ZNF217 and reduced
survival duration in human ovarian cancer.
Materials and Methods
QuMA
During QuMA, test and reference loci containing (CA)n repeats were
amplified using PCR in the presence of TaqMan probe homologous for a
(CA)n repeat. The amount of FAM fluorescence in each reaction liberated by
the exonuclease degradation of the TaqMan probe during PCR amplification
was measured as a function of PCR cycle number using an ABI 7700 Prism
(PE Biosystems, Foster City CA; Refs. 6–9). The number of PCR cycles (Ct)
required for the FAM intensities to exceed a threshold just above background
was calculated for the test and reference reactions. Ct values were determined
for three test and three reference reactions in each sample, averaged, and
subtracted to obtain Ct [Ct Ct (test locus) Ct (pooled reference)]. Ct
values were measured for each unknown sample [Ct (test DNA)] and for
samples from several unrelated, normal (calibrator) individuals [average
Ct (calibrator DNA)]. Relative copy number at each locus in the test
sample was then calculated as:
Relative DNA copy number 1 E Ct (1)
where Ct Ct (test DNA) Ct (calibrator DNA), and E PCR
efficiency. For simplicity, we used primers with PCR efficiencies of 85%
and calculated the relative DNA copy number as 2 Ct ( 2 when known to
be a diploid test sample, such as normal male DNA, or mouse knockout DNA
or with FISH analysis of mouse tumors).
To determine whether a QuMA measurement on a single sample was significantly
different from the mean of measurements made on samples from a number
of normal individuals, a TI was calculated using the mean and SD of Ct values
from each marker and the pooled reference according to the following formula:
TI 2 {SD (all markers) 2.28}) ( 2 for mouse leukemia DNAs) (2)
where 2.28 was a two-sided tolerance limit factor assuming a normal distribution
for a total of n 140 mouse loci (10). The measured relative copy
number of a normal sample will be within 2 TI 95% of the time with 95%
confidence. Thus, individual measurements outside this range were considered
significantly different from normal.
PCR
PCR was conducted in triplicate with 50-l reaction volumes of 1 PCR
buffer A (PE Biosystems), 2.5 mM MgCl 2, 0.4 M each primer, 200 M each
deoxynucleotide triphosphate, 100 nM probe, and 0.025 unit/l Taq Gold (PE
Biosystems) with 1–5 ng of genomic DNA. A large master mix of the
above-mentioned components was made for each experiment and aliquoted
into each optical reaction tube. Each primer set (5–10 l volume) was then
added, and PCR was conducted using the following cycle parameters: 1 cycle
of 95°C for 12 min and 40 cycles of 95°C for 20 s, 55°C for 20 s, 72°C for 45 s.
Analysis was carried out using the sequence detection software supplied with
the ABI 7700 (PE Biosystems). The Ct values for each set of three reactions
were averaged for all subsequent calculations. Pooled variance for all sets of
PCR triplicates was 0.018 (n 288), indicating sufficient statistical power
with this level of intra-assay variation to detect a difference of 0.25 cycle
between samples with greater than 95% confidence.

Oligonucleotides
PCR primer sequences for microsatellite loci were obtained from either the
Whitehead Institute Massachusetts Institute of Technology Center for Genome
Research 3 or Washington University (St. Louis, MO). 4 Primers were synthesized
by Life Technologies, Inc. (Gaithersburg, MD) or Integrated DNA Technologies
(Coralville, IA). The reference pools contained primer pairs for six or seven
different loci. For human analyses, loci were chosen in regions of the genome that
3 World Wide Web address: www.genome.wi.mit.edu.
4 World Wide Web address: www.genlink.wustl.edu.
QUANTITATIVE MICROSATELLITE ANALYSIS
Table 1 Primers used in QuMA studies
Forward primer Reverse primer
Human primers for
D8S258 CTGCCAGGAATCAACTGAG TTGACAGGGACCCACG
D15S126 GTGAGCCAAGATGGCACTAC GCCAGCAATAATGGGAAGTT
D17S849 CAATTCTGTTCTAAGATTATTTTGG CTCTGGCTGAGGAGGC
DXS453 CACACTGCATTAAATCCTCG CAAGTTACCCTACCTGCCTC
DXS1052 GCCATGTGTCAAGGCTGATT TAACCTCTCTCGCTCGCTCT
DXS1196 CTAAATTCTCCTCCACCGTG TTTCCAGAGCAGATTTTCAGT
D20S120 TTTTACTAAGGAGACTCAACAGGG TTGCACAATGCCTGGAA
D20S183 GCACATAAAACAGCCAGC CCGGGATACCGACATTG
D20S211 TTGGAATCAATGGAGCAAAA AGCTTTACCCAATGTGGTCC
D20S876 ATCATTTAGGGATTAAGTTCCTAC ATCACGCCACTGCACT
D20S894 GAAGCACCCTGTGGAGTTA CAATCCTGCAAAAAAGTCA
D20S902 GGACGTTCACACTTTATGCC GAGGATGCTTGCCATTTTC
D20S913
Mouse primers for
CTGCTCTAAGGTCAGAGTGATG CGATATGTAGGTTTACAAGGTTCC
D2MIT62 GGATACCGTTTGGAAAGTAAACC GCAAGAAGCACAGGAGGC
D2MIT63 GCAGTCTACCAGGAGCAACC TGGATGTAGGCATGTGCCT
D2MIT65 CTGAAAAAGGAAACAAGGTTGG CCCACTCTTTAGACAGAGGGG
D2MIT57 CCTCACAACTATGTCAGGTAATGG GGATGAGGGATTAAAAATAGATGC
D2MIT58 ATAGTTGGTTTTTGTTGCTATTGC TGCATTTGCAACATACCCC
D2MIT15 ATGCCTTAGAAGAATTTGTTCCC CTTGAAAAACACATCAAAATCTGC
D2MIT100 GTGTTCCTAAGGTTGTATTTTGGC GAAATTTGACAATTGCTAGGTGC
D2MIT107 GGGAGTGAAGCCAGCATAAG AACTGACTGAGTTTCAAAGTGCC
D2MIT112 ACTGTGTGCTTTTGTTTTGGG CAACACTATGGAGGCAGGGT
D2MIT142 AGCAAGGCATAAGGAGACCA GGGTGGCTCTATTAGGCACA
D2MIT175 ATGACAAACAAGAAATAGGAAGGC CATGTGCTTGAGCATGCAC
D2MIT224 GGAACTACTAACTAGGTGAATTCACTG CTTGAGAATGTTATGCTAGGAAGG
D2MIT244 AGGGCTCTGCTCATGCAC ACCACCTGTATCATGGACTTCA
D2MIT262 ACTCTCATGTGTGCCATTATGC TGTTAGCCTCTCTGCTTCTGC
D2MIT304 AAGCAGGTGTCGCTGATTG AGAAGATGGACCGAGGGG
D2MIT337 GATGCAATTCCTTCCAGCAT GAACCACTGTGTGATCGTCTATG
D2MIT339 TTGCACCAGGTTCTCAGAGA TAAGTGTTGCAGCATTTGTGC
D2MIT356 TGCAGTGCCAGATGTGTGT TCGACTCCTGTCTCTCTCAGG
D2MIT373 AGTGATTGAGGAAGACGTTTTACC GAACTTGCTCTGCCCAGTTC
D2MIT395 AGGTCAGCCTGGACTATATGG AGCATCCATGGGATAATGGT
D2MIT415 AGACCCGAGTCCCAAAAGTT CTGGCTCTCTGAGTCCGTTC
D2MIT419 ACCAGGAACCTGAGACTAAATATCC GGGGATAAATAAAAATAAACAAGAATC
D2MIT423 TAGAATGTTTGTCTGTTGATGAATATG CAGAGAATGGGCAAGTACAGC
D2MIT436 ATCCACGAGTGTCTTTTCCC TGTTAAGTTTTGGAGAAGAGTAAAAGG
D2MIT447 CATGTGCCATGGTACAAAGC TGGCTTGTTTCAGATAGCACA
D2MIT493 GTCTCTACCTGAGTTTCCATCACA TCCCGAGTTGTCCCTCTATG
D2MIT9 GTCTGCACTCTCACCAGCAA CAGCTTGAAATGCCTTTGAG
D3MIT12 TAGACCAATCTTGGGAGTGTCC GGAAAAGCATAAGAAACAACCG
D7MIT7 ACTCAAAGGTTGTCCTGGCA TGGTAGTGGTGGCTNCGGTG
D11MIT23 AAAGCAGAGAAGTCTCACCCCT GCACACAGTCATGCAGCAC
D12MIT10 ATCGTGGGAACAAGACCATC AGAATTCATGCTTTGCATTGG
D13MIT184
Human reference pool primers for
ACGCTAGGAAGTGGCTCAAA ACATGTGTCAGTCTTCGTTCTCA
D1S2868 AGGTATAATCTGCAATAAAAAACTT AAAGTAAAACAATATGAAGCCAC
D2S385 AGCTGTCAGTAGAAATAAGCAGAGA TCAATAACACGCCAAAAGAC
D4S1605 CATTCTAGTAGTTATTGGCTTATCC CAGTTGCTTGATACCTATATTTTTC
D5S478 CCGTTTGCACATTTGAGGCT CTGAAAAAGAAGGCAACGTC
D5S643 TGGGCGACAGAGCCATC TGTGGTGTGCCATTTATTGACT
D10S586 TATTATACTCCAGCCGGGG GGAGACTATTTACTTTGTGTCCTTG
D11S1315 AAAGGCACAAAAACTAAAACTCTGG CCGTCAGTGTGATAAAAGCCAG
D11S913 CTAGATTTCTGAAACTCAAATGCA TAGGTGTCTTATTTTTTGTTGCTTC
D12S1699 ATCACCTCATGCCTGTTAGG GTGTTTCGTTCACATCCTGG
D14S988 TGGTGATTGGATATCACTGGT ATGTTATGTAAGGTTTTGTTTTGTT
D21S1904 ATGAGTTCAGTGTTTCATGGACATC AGCAAGATTACTGTCTGGTTTCCC
D22S922
Mouse reference pool primers for
GTATCTTGATGGTGGTGTTGG TTTCCTCAGTTTTACCTGTGCT
D1MIT64 AGTGCATTATGAAGCCCCAC TCAAATTTTAAAACAACCCATTTG
D2MIT175 ATGACAAACAAGAAATAGGAAGGC CATGTGCTTGAGCATGCAC
D3MIT12 TAGACCAATCTTGGGAGTGTCC GGAAAAGCATAAGAAACAACCG
D12MIT10 ATCGTGGGAACAAGACCATC AGAATTCATGCTTTGCATTGG
D13MIT250 ACACTCATTTCCATGCACGA AGGTCCTCAAATCTCACAAGTAGG
D14MIT5 CACATGAACAGAGGGGCAG GTCATGAAGTGCCCACCTTT
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usually did not show alterations in human breast tumors (human pool 1, D4S1605,
D5S478, D11S913, D12S1699, D14S988, D21S1904, and D22S922) or ovarian
tumors (human pool 2, D1S2868, D2S385, D4S1605, D5S643, D10S586, and
D11S1315) when analyzed by CGH (11). The murine reference loci were
D1MIT64, D2MIT175, D3MIT12, D12MIT10, D13MIT250, and D14MIT5.
Primer sequences for the test and reference loci analyzed in this study are listed in
Table 1. The TaqMan CA-repeat fluorogenic probe consisted of the following
sequence: 5-FAM-TGTGTGTGTGTGTGTGTGTGT-6-carboxy tetramethyl
rhodamine-3. ZNF217 primers and probe were as reported previously (12). Both
probes were purchased from PE Biosystems.

Fig. 1. QuMA and FISH analyses of human
chromosome 20q copy number in breast cancer cell
lines using precisely mapped BACs and SSRs (12),
plus BAC clone B4104 and P1 clone P4120. 5 a,
QuMA measurements of relative copy number for
breast cancer cell lines are shown as symbols connected
by lines. MCF-7 values are shown as
connected by solid lines. SKBR3 values are shown
as f connected by dashed lines. FISH measurements
of relative copy number are shown as horizontal
lines whose lengths indicate the physical
extent of the BAC used as probes. MCF-7 values
are shown as solid lines. SKBR3 values are shown
as gray lines. These cell lines show a moderate to
high level of amplification. b, QuMA measurements
of relative copy number for breast cancer
cell lines are shown as symbols connected by lines.
HS578T values are shown as f connected by long
dashed lines. HBL100 values are shown as
connected by short dashed lines. MDA361 values
are shown as Πconnected by solid lines. FISH
measurements of relative copy number are shown
as horizontal lines whose lengths indicate the physical
extent of the BACs used as probes. MDA361
values are shown as solid lines. HBL100 values are
shown as gray lines. HS578T values are shown as
vertically hatched horizontal lines. These cell lines
show normal to low-level copy number increase.
FISH
Murine spleen cells on microscope slides fixed using methanol-acetic acid
were analyzed using dual-color FISH with BAC probes as described previously
(13). Hybridization domains of each color were enumerated in at least
100 metaphase or interphase nuclei for each analysis. BAC clones containing
SSRs D2MIT356 (BAC356), D2MIT62 (BAC62), and D2MIT415 (BAC415)
were selected from a BAC library as described previously (14). DNA from
each BAC clone was extracted and purified using a midi-prep column according
to manufacturer’s recommendations (Qiagen, Santa Clarita, CA) and
labeled by nick translation to incorporate dCTP FITC or Texas Red (13).
Specimens
Murine. Two inbred strains of mice were evaluated: (a) a FVB knockout
mouse (designated M11) that lacked a 700-kb segment of chromosome 11
(kindly provided by Dr. E. Rubin; Lawrence Berkeley National Laboratory,
Berkeley, CA); tail DNA samples were obtained from M11 / , M11 / , and
M11 / FVB mice; and (b) 3-month-old SJL mice (Jackson Laboratories, Bar
Harbor, ME) exposed to 3 Gy of ionizing radiation from a Cs 137 irradiator.
Spleen and marrow samples were collected for DNA isolation after 7–9
months.
Human. All specimens were collected and used with the approval of the
Committee on Human Research at the University of California, San Francisco.
These specimens included: (a) blood samples collected from normal, healthy,
male and female donors for analysis of X chromosome copy number; (b)
ovarian tumor samples collected as described by Suzuki et al. (15); and (c)
human breast cancer cell lines HBL100, MDA-MB-361, HS578T, SKBR3,
and MCF-7 obtained from American Type Culture Collection (Manassas, VA)
and cultured as recommended by the supplier.
5 Collins, C., unpublished observations.
QUANTITATIVE MICROSATELLITE ANALYSIS
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DNA Isolation
DNA was extracted from either tail clippings or spleens of mice and
processed with a Puregene DNA extraction kit (Gentra Systems, Minneapolis,
MN) used according to the manufacturer’s purification recommendations.
DNA was extracted from human blood specimens and cell lines using a Wizard
Genomic DNA purification kit from Promega (Madison, WI). Ovarian tumor
DNA was isolated as described previously (15).
CGH
CGH was performed on DNA from three SJL mice as described by Kallioniemi
et al. (16). Mouse metaphase chromosome spreads were prepared
from phytohemagglutinin-stimulated murine peripheral blood lymphocytes of
a normal C57BL mouse. Image analysis was conducted as described by Piper
et al. (17).
Results and Discussion
Validation
Amplification. We used QuMA to measure the levels of amplification
of SSR loci mapped to 20q13.2 and the putative oncogene
ZNF217 in five breast cancer cell lines that had been assessed previously
using FISH (11, 18). Fig. 1 shows good concordance between
the two methods. Cell lines HBL100, MDA-MB-361, and HS578T
show only modest increases (less than five copies) in DNA copy
number, whereas SKBR3 and MCF-7 show high-level amplifications
(up to 40 copies). However, the levels of amplification measured
using FISH and QuMA differ by as much as 2 at some loci. We
speculate that this is because the extent of the genome interrogated
differs between the two methods. QuMA assesses copy number at a

specific 200-bp wide locus, whereas FISH measures copy number
using an 100-kb probe. The number of hybridization signals generated
using FISH will vary in an uncertain manner if the level of
amplification changes across the extent of the region covered by the
probe. In addition, copy number is difficult to measure in interphase
nuclei using FISH when the level of amplification is high. On the
other hand, FISH is less sensitive to the presence of admixed normal
cells. The level of aneusomy in these cell lines may also contribute to
the difference because the reference loci may be elevated in copy
number, resulting in underrepresentation of the absolute DNA copy
number. Thus, analysis using both methods may be appropriate in
cases of high-level amplification or aneusomy.
Single Copy Number Changes. We assessed the ability of QuMA
to detect monosomy by comparing copy number measurements on the
X chromosome in human males with copy number at three autosomal
loci and by analyzing copy number in a region of chromosome 11
deleted in a mouse knockout experiment. QuMA analyses in the
human showed a relative copy number of 1.08 0.035 for three
autosomal loci (D8S258, D15S126, and D17S849) in male DNA and
0.46 0.06 for the three nonpseudoautosomal X chromosome loci
(DXS453, DXS1052, and DXS1196). QuMA measurements of relative
copy number at D11MIT23 in a 700-kb segment of chromosome
11 deleted by homologous recombination 6 and at six autosomal loci
(D1MIT64, D2MIT175, D3MIT12, D12MIT10, D13MIT250, and
D14MIT5) were 0.59 0.06 and 1.03 0.13, respectively. Both studies
clearly show that QuMA is sufficiently sensitive to distinguish one copy
from two copies.
To further define the sensitivity of QuMA, we mixed DNA from a
chromosome 11 knockout mouse with increasing amounts of DNA
from normal FVB mice. QuMA was performed on these DNA mixtures
at D11MIT23, D2MIT175, and D13MIT250. Fig. 2A shows that
QuMA was able to distinguish the reduced copy number at
D11MIT23 even in the presence of 30% of normal DNA. Specifically,
QuMA showed a 90% chance of detecting a difference between one
copy and two copies, with 95% confidence under these conditions.
Thus, QuMA should be able to detect the loss of one allele in tumor
samples contaminated by this amount of normal DNA.
Demonstration Applications
Deletion Analysis in Tumors in Inbred Mice. The SJL mouse
strain has been shown to develop acute leukemias at high frequency
when irradiated. Recurrent allelic imbalance of an 30-cM region of
chromosome 2E has been reported in tumors in F 1 crosses of these
mice (19, 20). We used QuMA to assess deletions at 27 loci along
chromosome 2 using spleen DNA from eight irradiated SJL mice. Six
of the eight spleens were comprised mostly of leukemic cells. Fig. 2B
shows that these analyses define an 23-cM-wide region of common
deletion between D2MIT9 and D2MIT337. The QuMA results are
consistent with those obtained using FISH and CGH. CGH analysis of
DNA from spleens from two irradiated mice with leukemia (mice
2.4B and 11.2) and one irradiated mouse without leukemia (mouse
2.1B) showed chromosome 2 copy number losses in the two leukemic
mice and no loss in the nonleukemic mouse. FISH analyses of spleens
from two other irradiated mice with leukemia (mice 2.5 and 12.2) and
one mouse without leukemia (mouse 8.5) were performed using BAC
clones mapped to chromosome regions 2A(BAC356), 2E(BAC62),
and 2H(BAC415). For the nonleukemic mouse (mouse 8.5), the
2A:2E copy number ratio was 0.92, whereas in leukemic mice (mice
2.5 and 12.2), the ratios were 1.67 and 1.89, respectively. The 2A:2H
ratios for the same mice were both 1.02, indicating that these mice
6 Rubin et al., unpublished observations.
QUANTITATIVE MICROSATELLITE ANALYSIS
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Fig. 2. A, DNA mixing experiment in which normal SJL DNA was added with
hemizygous chromosome 11 knockout DNA in increments of 10%. DNA copy number
was measured at marker D2MIT23 (, experiment 1; f, experiment 2) and is compared
with markers D2MIT65 (E, experiment 1; F, experiment 2) and D13MIT184 (‚,
experiment 1; Œ, experiment 2). B, leukemic and normal mice were measured for DNA
copy number using QuMA at 27 loci on chromosome 2 and 3 loci on other chromosomes.
Results for six leukemic mice show a common region of deletion between markers
D2MIT9 and D2MIT337 (23 cM).
have an interstitial deletion of in the region of 2E. These results
demonstrate the utility of QuMA for high-resolution mapping of the
extent of physical deletions in inbred mouse strains, obviating the
need for expensive and time-consuming backcrossing.
Reproducibility. To assess the reproducibility of QuMA, 13 markers
were measured three times on four irradiated mice. The average

Fig. 3. Kaplan-Meier survival curves for 60 patients with and without DNA copy
number gains at ZNF217 in chromosomal band 20q13.2, as detected by QuMA. a,
survival of all patients with ZNF217 gains. b, survival of patients with late-stage tumors.
relative copy number for the disomic loci was 2.00 0.26 (n 123),
with 5 of 123 measurements outside the TI (three above the cutoff
values and two below the cutoff values). The average relative copy
for monosomic loci was 0.94 016 (n 30), with all values outside
the TI.
Assessment of Prognostic Markers in Human Tumors. Amplifications
of several regions of chromosome 20q occur frequently in a
broad range of human cancers including ovarian cancer (11). Several
putative oncogenes have been identified in these regions including the
putative zinc-finger transcription factor ZNF217 (12). Amplification
of this gene has been associated with reduced survival duration in
human breast cancer (21). We used QuMA to test the association
between ZNF217 copy number increase and reduced survival for 60
ovarian cancer patients. The Kaplan-Meier survival curves for patients
with or without increased copy number of ZNF217 in Fig. 3 show that
increased copy number of this gene was significantly associated with
reduced survival duration in late-stage tumors and all tumors. This
study demonstrates the utility of QuMA for rapid detection of prognostic
copy number changes in human cancers.
Conclusion
We have demonstrated the utility of QuMA for rapid measurement
of relative DNA copy number in the genomes of mouse and human
tumors. QuMA distinguishes between increases and decreases in copy
number and takes advantage of the large number of SSRs that have
already been mapped and for which PCR primers are conveniently
available. Of course, it does not detect events leading to loss of
heterozygosity or other abnormalities that do not alter genome copy
number. The use of a single TaqMan probe for all test and reference
loci makes the method cost effective. The use of a “pooled reference”
reduces the possibility that loss/gain of a single reference marker
might lead to erroneous assignment of copy number at a test locus.
However, the pooled reference should be designed to include loci that
are not frequently altered in copy number in the system under study
and may not provide a useful reference for determining absolute copy
number in highly aneusomic tumors. Published CGH and LOH anal-
QUANTITATIVE MICROSATELLITE ANALYSIS
5409
yses may be used to guide the selection of reference loci. QuMA is
advantageous because it does not require that the SSRs be polymorphic.
This is particularly helpful in high-resolution analyses of the
extent of regions of genome copy number abnormality in humans and
inbred mouse tumors and for assessment of the presence of prognostic
copy number abnormalities in individual tumors.
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