DNA was extracted from blood and 10-μm frozen tumor sections
using standard procedures.
18 A 5-μm section from each
tumor was stained with hematoxylin and eosin to confirm the histology
and to ensure that the specimen was composed of at least 90% tumor
cells.
Fluorescence microsatellite assays and analysis on an automatic
sequencer were used to detect LOH. Microsatellite markers were chosen
from a mapping set (Linkage Mapping Set V2.0; PE-Applied Biosystems,
Warrington, UK). Seven markers were chosen, located within or close to
known or putative TSGs: D3S1300 (3p14.2; intragenic FHIT),
D3S1304 (3p26-p25; VHL), D9S161 (9p21; CDKN2A/p16), D9S157 (9p23-p22), D13S171
(13q12.3-q13; BRCA2), D13S153 (13q14.2; intragenic RB1), and D17S2179E (17p13; intragenic TP53).
These seven microsatellites were amplified simultaneously in multiplex
reactions. The reaction mixtures contained the microsatellite primers
at various concentrations (80–145 nM), buffer (GeneAmp Buffer II;
PE-Applied Biosystems), 350 μM dNTPs, 2.66 mM
MgCl2, and 3.5 U polymerase (AmpliTaq Gold; PE-Applied Biosystems). Thermocycling conditions were as follows:
initial denaturation for 12 minutes, followed by 30 cycles of 94°C
for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, with
a final extension step at 72°C for 20 minutes. PCR products were
diluted 2.5-fold in deionized distilled (dd)H2O
and mixed with loading buffer (size marker ROX-350, dextran blue, and
formamide in a ratio of 1:1:5). After denaturation at 95°C for 5
minutes, samples were analyzed on an automatic sequencer (Prism 377,
using Genescan and Genotyper software; PE-Applied Biosystems).
For heterozygous samples a reduction of at least 23% (allelic
imbalance factor [IF] of 0.77) in the peak area of one allele in the
tumor (normalized against the retained allele) was used to score
LOH.
19 In each case the IF was determined by calculating
the ratio of alleles for both the normal (N) and tumor (T) sample, and
then the tumor ratio was divided by the normal ratio: T1:T2/N1:N2. If
the value obtained was greater than 1.00, the reciprocal
1/(T1:T2/N1:N2) was used (to give a range of 0.00 to
1.00).
20 21 Samples with 0.65 to 0.77 on initial analysis
were subjected to another assay and scored as LOH only if a second
result less than 0.77 was obtained.
19 The cut-off level
used (IF 0.77) was determined by calculating interassay variation using
normal DNA.
19 To assess reproducibility, 24 normal blood
DNA samples were subjected to multiplex assays, with each sample
repeated four times. Comparison of allele ratios of these normal
samples showed a SD of 0.076, indicating that the reaction-to-reaction
variation was reasonably small and that allelic imbalance carried by at
least 23% of the cells in the sample could be detected with an
extremely high confidence. This cut-off was also confirmed by mixing
normal and tumor DNA in various ratios.
Further microsatellite assays were undertaken using 10 chromosome 3p
and 3q markers (Research Genetics, Huntsville, AL): D3S1038
(3p26.1-p25.2), D3S1283 (3p25-p24.2), D3S1619 (3p24.2-p22), D3S1029
(3p21.3-p21.2), D3S1210 (3p14.1-p12), D3S1271 (3cen-q13), D3S1589
(3q21), D3S1605 (3q25.1-25.2), D3S1580 (3q27), and D3S1311 (3q27-qter).
An additional seven markers located on chromosome 9p23-p21 were also
analyzed: D9S156, D9S162, D9S1846, D9S1749, D9S1748, D9S1679, and
D9S171 (Research Genetics). Amplification products were analyzed on
silver-stained polyacrylamide gels,
22 and LOH was recorded
for informative markers if the intensity of a tumor allele was reduced
by at least 30% relative to the corresponding normal DNA. This change
in allele intensity was obvious visually (see
Fig. 3 ).