Recruitment and tumor samples
Between January 2004 and September 2005 all women undergoing primary debulking surgery for ovarian carcinoma were invited to participate in this study at the Vancouver General Hospital and British Columbia Cancer Agency in Vancouver, Canada. Women with mucinous and borderline tumors, and women who had received pre-operative chemotherapy were excluded. Pathology was reviewed by a single pathologist (CBG). Serous tumors were classified as low or high-grade as described previously ; all undifferentiated and clear cell carcinomas were considered high-grade. Endometrioid carcinomas were graded as grade 1, 2, or 3 according to the Silverberg grading system . 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 instructions from whole blood (germline analysis) or tumor samples (somatic analysis). RNA was isolated with Trizol (Invitrogen, Carlsbad, CA) according to standard protocols.
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)) , and BRCA2 (D13S260 (60°C), D13S171 (50°C), D13S267 (53°C), D13S217 (55°C)) . PCR products were electrophoresed 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 reduction of one allele in at least one marker. Microsatellite instability (MSI) was defined as the presence of novel alleles in the tumor DNA that were not present in normal DNA in at least one marker .
dHPLC mutation screening and mutation analysis
Screening for BRCA1/BRCA2 mutations was performed using denaturing high performance liquid chromatography (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 . 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 available 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 multiple exon deletions or duplications, multiplex ligation-dependent probe amplification analysis (MLPA) kits SALSA P002 BRCA1 and SALSA P087 BRCA1 (MRC Holland, Amsterdam, NL) were used according to manufacturer 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 .
BRCA1 and FANCF promoter hypermethylation analysis
The BRCA1 methylation status of each tumor was assessed using a technique similar to the MethyLight assay described previously . 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 MYOD1 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 fluorescent signal. The sequences of the primers used to amplify and detect methylated BRCA1 promoter region were 5'-TAGAGTTTCGAGAGACGTTTGGTTT-3' (forward primer) and 5'-CGCTTTTCCGTTACCACGA-3' (reverse primer). The primers for MYOD1 were 5'-CCA ACTCCA AATCCCCTC TCTAT-3' (forward primer) and 5'-TGATTAATTTAGATTGGGTTTAGAGAAGGA-3' (reverse primer). The amount of methylated DNA (PMR, percentage of methylated reference)  at the BRCA1 locus was calculated by dividing the BRCA1: MYOD1 ratio of a sample by the BRCA1: MYOD1 ratio of CpG methylated Jurkat genomic DNA (New England Biolabs, Ipswich, MA) and multiplying by 100. Reactions using CpG methylated Jurkat genomic DNA were used to normalize for any difference 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 . 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 gene of interest are gained relative to MYOD1 in any given sample. PCR experiments were carried out in a volume of 10 μL with 384-well plates and an Applied Biosystems 7900 HT Sequence Detector (Applied Biosystems, Foster City, CA). The fluorescence signal of the quantitative methylation-specific PCR was generated by SYBR Green I. Samples (10 ng bisulfite-treated DNA) were run in triplicate containing 5 μL SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and 5 pmol of each forward and reverse primer. Bisulfite-converted CpG methylated Jurkat Genomic DNA (New England Biolabs, Ipswich, MA) served as a positive control and was used to generate a standard curve to quantify the amount of fully methylated promoters in each reaction. PCR amplification was done by means of the following procedure: 95°C for 15 minutes, followed by 40 cycles at 95°C for 15 seconds, 62°C for 1 minute. A subsequent dissociation curve analysis checked the specificity of products. FANCF promoter hypermethylation was assessed using a HpaII digest assay and methylation-specific PCR protocol previously reported by Taniguchi et al .
Extracted RNA was treated with DNAse I (Invitrogen, Carlsbad, CA) prior to creating cDNA using random hexamer priming and MMLV reverse transcriptase (Invitrogen, Carlsbad, CA). Applied Biosystems Taqman primer/probe kits (Hs00173233_m1 (BRCA1), Hs00609060_m1 (BRCA2), Hs01920652_s1 (PTEN), Hs00907966_m1(PIK3CA)) were used to quantify mRNA expression levels using real-time qRT-PCR  and an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA). Relative gene expression quantification was calculated according to the comparative Ct method using human 18S ribosomal RNA (Applied Biosystems, Foster City, CA) and commercial RNA controls (Stratagene, La Jolla, CA). Relative quantification was determined as follows: 2-(ΔCt sample-ΔCt calibrator). Ratios (tumor relative gene expression:average of all tumors) less than 0.7 or greater that 1.3 for were scored as decreased or increased mRNA expression, respectively.
The BRCA1 antibody Ab-1 (Oncogene, 1:50 dilution) was used and antigen retrieval was performed in 1× EDTA buffer (pH 8.0) by microwaving for 2 minutes, and then boiling in a waterbath for 30 minutes. Endogenous peroxide activity was blocked with 3% hydrogen peroxide and then sections were incubated with 2.5% normal horse blocking serum. Following incubation with the primary antibody, the Vector Laboratories (Burlingame, CA) ImmPRESS kit was used according to the manufacturer's recommendations to visualize antibody complexes. Nuclear staining was assessed by CBG, who was blinded to all other BRCA analysis. Tumors were considered BRCA1 positive if greater than 1% of tumor nuclei showed staining. IHC was also performed with the following panel of previously validated antibodies using a Ventana (Tucson, AZ) automated immunostainer: p21 (Neomarkers, Fremont, CA, clone DCS-60.2, 1:100 dilution), p53 (Dako, Carpinteria, CA, clone DO-7, 1:400 dilution), and WT-1 (Dako, Carpinteria, CA, clone 6F-H2, 1:50 dilution). BRCA1 IHC was done on whole sections, while other IHC markers were assessed using sections from a tissue microarray constructed with two 0.6 mm cores per case.
Molecular Inversion Probe (MIP) Copy Number
The MIP copy number assay was done as described previously  with some modifications. Specifically, the current protocol is a modification of the Targeted Genotyping protocol commercialized by Affymetrix . Test DNA samples were diluted to16 ng/μl. Molecular inversion probes were annealed to DNA by mixing 4.7 μl of DNA (75 ng total), 0.75 μl of Buffer A, 1.1 μl of the 53 K molecular inversion probe pool (200 amol/μl/probe) and 0.045 μl of Enzyme A in a 384-well plate on ice. The reaction was incubated for 4 min at 20°C, 5 min at 95°C, then overnight at 58°C. Following annealing, 13 μl of Buffer A and 1.25 μl of Gap Fill Enzyme mix were added to each reaction and 9 μl of reaction volume was transferred to each of two wells in a 96-well plate. Molecular inversion probes were circularized with 4 μl of dNTP mix at 58°C for 10 min. Linear probes and genomic DNA were eliminated by addition of 4 μl of Exo Mix and incubation at 37°C for 15 min, followed by universal primer amplification for 18 cycles (20 sec at 95°C, 40 sec at 64°C, and 10 sec at 72°C). For labelling reactions, the product was further amplified for 10 cycles using labelled primers, then subjected to cleavage by HY Digest Mix at 37°C for 2 hours. The cleaved MIP products were mixed with Hybridization Cocktail, denatured, and hybridized to 70 K Universal Taq arrays at 39°C for 16 h (two arrays per sample). The overnight hybridized arrays were washed on a GeneChip® Fluidics Station FS450 and stained by SAPE at 5 ng/ml (Invitrogen).
Copy number estimation was obtained from the hybridization signals as previously described , with the following modifications: the color-separation step was omitted as the single color readout on two arrays prevented the spectral overlap that occurs with multi-color readouts, and Langmuir correction was performed instead of linear calibration of allele signals . Copy numbers over 3.0 were considered amplification events and copy numbers below 1.5 were considered deletion events.
Epigenetic BRCA1 loss was defined as having promoter hypermethylation accompanied by either low relative BRCA1 mRNA expression, negative BRCA1 IHC, or both low BRCA1 mRNA and negative BRCA1 IHC. Tumors without promoter hypermethylation and with positive BRCA1 IHC were not considered to have BRCA1 loss. Tumors with negative BRCA1 IHC without promoter hypermethylation were considered equivocal for BRCA1 loss. A chi-squared test or Fisher exact test was used to analyze categorical variables (MIP copy number, IHC) and a student's t-test was used to analyze continuous variables (RNA expression).