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mas were graded as grade 1, 2, or 3 according to the
Silverberg grading system [45]. Ethical approval was
obtained from the University of British Columbia Ethics
Board (#H02-61375 and #H03-70606).
DNA and RNA extraction
Cancer tissue was split with part stored at -80 degrees and
the facing tissue fixed in formalin and placed in paraffin
blocks. H&E sections were reviewed to ensure samples
consisted of > 70% tumor cells. DNA was extracted using
the Puregene DNA Purification Kit (Gentra Systems, Inc,
Wicklow, Ireland) according to manufacturer's instruc-
tions from whole blood (germline analysis) or tumor
samples (somatic analysis). RNA was isolated with Trizol
(Invitrogen, Carlsbad, CA) according to standard proto-
cols.
Loss of heterozygosity analysis
Somatic loss of BRCA1/BRCA2 in tumor tissue was
assessed for LOH using microsatellite markers for BRCA1
(D17S855   (60 C), D17S1185    (58 C), D17S1323
(56 C), and D17S1325 (56 C)) [46], and BRCA2
(D13S260 (60 C), D13S171 (50 C), D13S267 (53 C),
D13S217 (55 C)) [9]. PCR products were electro-
phoresed in an ABI Prism 3100 Genetic Analyzer (Applied
Biosystems, Foster City, CA), and analyzed with Genescan
v3.1 software (Applied Biosystems, Foster City, CA). LOH
was defined as a complete or partial (  50%) signal reduc-
tion of one allele in at least one marker. Microsatellite
instability (MSI) was defined as the presence of novel alle-
les in the tumor DNA that were not present in normal
DNA in at least one marker [47].
dHPLC mutation screening and mutation analysis
Screening for BRCA1/BRCA2 mutations was performed
using denaturing high performance liquid chromatogra-
phy (dHPLC). Tumor DNA was mixed in a 3:1 ratio with
corresponding germline DNA for all tumors shown to
possess LOH to ensure that LOH did not mask somatic
mutations [48]. For example, with intratumoral LOH, and
mutation of the remaining allele, dHPLC screening would
give a false negative result. If the mutation is a germline
mutation it will be picked up on dHPLC screening of
germline DNA, however, a somatic mutation would be
missed in both tumor and germline DNA without DNA
mixing. PCR primers and conditions were developed by
the Royal Melbourne Hospital (Australia) and are availa-
ble on request. PCR primers were used to amplify each
exon of BRCA1 (24 exons) and BRCA2 (26 exons). All
exons with abnormal dHPLC profiles were PCR amplified
and bi-directionally sequenced to identify mutations
using ABI BigDye terminator v3.1 cycle sequencing kit
(Applied Biosytems, Foster City, CA) and an ABI Prism
3100 Genetic Analyzer (Applied Biosystems, Foster City,
CA).

Multiplex Ligation-dependent Probe Amplification
(MLPA) screening
For the identification of germline BRCA1 single and mul-
tiple exon deletions or duplications, multiplex ligation-
dependent probe amplification analysis (MLPA) kits
SALSA P002 BRCA1 and SALSA P087 BRCA1 (MRC Hol-
land, Amsterdam, NL) were used according to manufac-
turer directions. A reduction or increase in RPA values to
<0.7 or >1.3 was considered an indication of a deletion or
a duplication, respectively [49].
BRCA I and FANCF promoter hypermethylation analysis
The BRCA1 methylation status of each tumor was assessed
using a technique similar to the MethyLight assay
described previously [50]. Briefly, 500 ng of sample DNA
was subjected to sodium bisulfite modification using the
EZ DNA Methylation-Gold Kit, as recommended by the
manufacturer (Zymo Research, Orange, CA). After
bisulfite treatment, DNA was amplified using real-time
PCR with oligonucleotide primers complementary to a
region of the MYOD 1 promoter that did not contain any
CpG dinucleotides but did contain non-CpG cytosines to
ascertain the amount of converted input templates in each
sample. Hypermethylation of the BRCA1 promoter was
then examined by real-time PCR amplification of
bisulfite-modified DNA using oligonucleotide primers
specific for a fully methylated bisulfite-converted portion
of BRCA1 promoter such that only CpG islands that were
methylated at every CpG dinucleotide interrogated by the
primers and probes would be amplified and generate flu-
orescent signal. The sequences of the primers used to
amplify and detect methylated BRCA1 promoter region
were 5'-TAGAGTITCGAGAGACGTITGGTIT-3' (forward
primer) and 5'-CGCTITTCCGTTACCACGA-3' (reverse
primer). The primers for MYOD1 were 5'-CCA ACTCCA
AATCCCCTC TCTAT-3' (forward primer) and 5'-TGAT-
TAATITAGATTGGGTITAGAGAAGGA-3'             (reverse
primer). The amount of methylated DNA (PMR, percent-
age of methylated reference) [51] at the BRCA1 locus was
calculated by dividing the BRCA1: MYOD1 ratio of a sam-
ple by the BRCA1: MYOD1 ratio of CpG methylated Jur-
kat genomic DNA (New England Biolabs, Ipswich, MA)
and multiplying by 100. Reactions using CpG methylated
Jurkat genomic DNA were used to normalize for any dif-
ference in amplification efficiencies between BRCA1 and
MYOD1. The PMR serves as an index of the percentage of
bisulfite converted input copies of DNA that are fully
methylated at the primer hybridization sites. The PMR
values obtained by MethyLight were dichotomized at 4
PMR for statistical purposes as described previously [51].
Samples containing 4 PMR or higher were designated as
methylated, whereas samples containing less than 4 PMR
were designated as unmethylated. It is important to note,
however, that the PMR may be >1 if copies of MYOD1 are
deleted relative to the gene of interest, or copies of the

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BMC Cancer 2008, 8:17