Telomeric Allelic Imbalance Indicates Defective DNA Repair and Sensitivity to DNA-Damaging Agents

Cell lines carrying BRCA1 or BRCA2 mutations are more sensitive to killing by the platinum salts cisplatin and carboplatin than are wild-type cells (1, 2). Breast and ovarian cancers in patients carrying BRCA1 or BRCA2 mutations are likewise sensitive to platinum-based chemotherapy (3, 4). The majority of breast cancers arising in women with a germline BRCA1 mutation lack expression of estrogen and progesterone receptors or amplification of the HER2-neu gene (“triple-negative”). BRCA1-related breast cancers share a number of phenotypic characteristics with sporadic triple-negative breast cancer [TNBC (5–7)]. Both tumor types share a common pattern of genomic abnormalities and have high global levels of chromosomal aberrations, including allelic imbalance (AI), the unequal contribution of maternal and paternal DNA sequences with or without changes in overall DNA copy number (8–10). Because they have in common genomic aberrations suggesting a shared lesion in genomic integrity control, it is reasonable to posit that sporadic TNBC that has accumulated high levels of AI might share the sensitivity to platinum-based chemotherapy that characterizes BRCA1-associated cancer.

These observations prompted a clinical trial, Cisplatin-1, in which 28 patients with operable TNBC were treated preoperatively with cisplatin monotherapy. Preoperative treatment in the Cisplatin-1 trial resulted in greater than 90% tumor reduction in 10 of 28 (36%) patients, including pathologic complete response (pCR) in 6 women, 2 of whom had BRCA1-associated cancers (11). A second trial, Cisplatin-2, accrued 51 patients with TNBC who received the same preoperative cisplatin regimen as those in Cisplatin-1, but in combination with the angiogenesis inhibitor bevacizumab (12).

The response rate in Cisplatin-2 was similar to that in Cisplatin-1. In the second trial, a greater than 90% tumor reduction was observed in 17 of 44 women (39%) completing treatment. In Cisplatin-2, 8 patients carried a germline BRCA1 or BRCA2 mutation, of which 4 patients achieved a pCR or near pCR to the cisplatin–bevacizumab regimen. In both trials, all patients had research sequencing to determine their germline BRCA1 and BRCA2 status. We compared the number of various chromosomal abnormalities including AI present in tumor biopsies obtained before therapy to pathologically determined tumor response to cisplatin, alone or in combination with bevacizumab, assessed by examination of the posttreatment surgical specimen.

Chromosomal abnormalities such as regions of AI, other than those resulting from whole chromosome gain or loss, might result from improper repair of DNA double-strand breaks during tumor development. If so, then a genome-wide count of abnormal chromosomal regions in tumors may indicate the degree of DNA repair incompetence, independent of knowledge of any specific causative DNA repair defect. We hypothesized that the number of chromosomal regions of AI in tumors would predict sensitivity to drugs that induce DNA cross-links such as cisplatin.

We first sought associations between various measures of subchromosomal abnormalities and sensitivity to cisplatin in breast cancer cell lines and found the most accurate predictor to be AI extending to the telomeric end of the chromosome (N_tAI). Finally, we tested whether N_tAI was associated with treatment response in patient tumor samples in the Cisplatin-1 and Cisplatin-2 TNBC trials and in The Cancer Genome Atlas (TCGA) public data set of serous ovarian cancer, a cancer routinely treated with platinum-based therapy. In an effort to understand more about the processes leading to telomeric AIs, we mapped the location of their breakpoints and observed a striking association of these breakpoints with regions of the genome that are difficult to replicate—common copy number variants (CNV). Furthermore, a subset of high N_tAI tumors displays low BRCA1 mRNA levels. These observations begin to suggest models of how telomeric AI may occur.

We obtained single-nucleotide polymorphism (SNP) genotype array data from the Wellcome Trust Sanger Institute for a set of established BRCA1 wild-type breast cancer cell lines for which we had determined cisplatin sensitivity [Fig. 1A (13)]. Allele copy number was determined from the SNP array data and AI detected by the use of allele-specific copy number analysis of tumors, or ASCAT [Supplementary Fig. S1 (10)]. We tested for an association between the IC₅₀ values for cisplatin and each of 3 summary measures of chromosomal alteration: the number of chromosome regions with AI (N_AI; Supplementary Fig. S2A), the number of regions with copy number gains (N_Gain; Supplementary Fig. S2B), and the number of regions with copy number loss (N_Loss; Supplementary Fig. S2C). None of these measures was correlated with cisplatin sensitivity in the cell lines.

Known defects in DNA double-strand break repair, including loss of BRCA1, cause the spontaneous formation of triradial and quadriradial chromosome structures, which are cytologic indications of aberrant chromosome recombination (14–16). The resolution of these chromosome rearrangements at mitosis can result in large regions of AI and/or copy number changes extending from the cross-over to the telomere (15, 17). More generally, several error-prone repair processes potentially used by cells with defective DNA repair cause chromosome cross-over or copy choice events that result in allelic loss or copy number change extending from the site of DNA damage to the telomere.

We therefore looked for an association between cisplatin sensitivity and the number of contiguous regions of AI, copy gain, or copy loss that either extended to a telomere and did not cross the centromere (telomeric regions) or did not extend to a telomere (interstitial regions; Supplementary Fig. S1, Fig. 1B, and Supplementary Fig. S3). The number of regions of telomeric AI was the only summary genomic measure that was significantly associated with cisplatin sensitivity in the breast cancer cell lines (r_s = 0.76, P = 0.011; Fig. 1B); the correlation between N_tAI and cisplatin sensitivity was stronger when the analysis was restricted to the TNBC lines (Fig. 1B, red circles; r_s = 0.82, P = 0.0499). A similar relationship was observed between N_tAI and cisplatin sensitivity as measured by GI₅₀ in a recently published study of breast cancer cell lines [r_s = 0.57, P = 0.0018; Fig. 1C (18)]. Of all the drugs tested in this study, N_tAI was most highly correlated to cisplatin sensitivity.

We then investigated whether the association between N_tAI in clinical tumor samples and cisplatin sensitivity was present in the Cisplatin-1 trial. Sensitivity was measured by pathologic response determined after preoperative treatment (11). Molecular inversion probe SNP genotype data from pretreatment tumor samples (n = 27) were evaluated by ASCAT to determine N_tAI. We compared tumors with a reduction of at least 90% in the content of malignant cells (cisplatin-sensitive) to tumors with limited or no response to cisplatin (cisplatin-resistant, defined by tumor reduction of <90%). Cisplatin-sensitive tumors had significantly higher levels of N_tAI (median 24 vs. 17.5, P = 0.047, Fig. 2A). We tested the ability of N_tAI to predict cisplatin response by calculating the area under the receiver operating characteristic (ROC) curve (AUC). ROC analysis showed that higher levels of N_tAI were associated with cisplatin sensitivity [AUC = 0.74, 95% confidence interval (CI) 0.50–0.90; Fig. 2B].

In the Cisplatin-2 trial, cisplatin-sensitive tumors (n = 9) had significantly greater N_tAI than -resistant tumors (n = 17, median 27 vs. 20, P = 0.019; Fig. 2C). N_tAI was also associated with response to cisplatin and bevacizumab by ROC analysis (AUC = 0.79, 95% CI 0.55–0.93; Fig. 2D). The association between N_tAI and cisplatin sensitivity remained significant when cases with BRCA1 or BRCA2 mutation were excluded and only BRCA normal cases were analyzed (P = 0.030 and P = 0.023 in Cisplatin-1 and Cisplatin-2, respectively). Therefore, in 2 separate preoperative trials in breast cancer, in which treatment sensitivity was assessed by a quantitative measure of pathologic response, N_tAI reliably forecast the response to cisplatin-based treatment.

To test whether the N_tAI metric indicates platinum sensitivity in cancers other than breast, we determined the association between N_tAI and initial treatment response in a TCGA cohort of serous ovarian cancer patients who had received adjuvant platinum and taxane chemotherapy (19). Among the ovarian cancers without mutation in BRCA1 or BRCA2 (wtBRCA), the platinum-sensitive tumors had significantly higher levels of N_tAI than did the platinum-resistant cancers (median 22 vs. 20, P = 0.036; Fig. 3) and N_tAI was predictive of treatment response by ROC analysis (AUC = 0.63, 95% CI 0.50–0.76, Supplementary Fig. S4). The ovarian cancers with somatic or germline mutation in BRCA1 or BRCA2 that were sensitive to platinum therapy had even higher levels of N_tAI (median = 26, P = 0.0017 and median 23.5, P = 0.037 vs. resistant wtBRCA, respectively, Fig. 3). All of the BRCA2-mutated cancers were platinum sensitive; however, 5 BRCA1-mutated tumors were resistant to platinum therapy yet appeared to have relatively high levels of N_tAI. Thus, high N_tAI is characteristic of serous ovarian cancer with known mutation in either BRCA1 or BRCA2; high N_tAI is also found in a subset of sporadic cancers without BRCA mutations, where it is predictive of platinum sensitivity.

To develop a better understanding of the processes leading to telomeric AI, we mapped the location of the chromosome breakpoints defining the boundary of the telomeric AI regions. We observed that many breakpoints were located in very close proximity to each other (Supplementary Fig. S5), suggesting a nonrandom distribution of DNA breaks causing telomeric AI. Recurrent chromosomal translocation breakpoints may be associated with regions of repeated DNA sequence that may cause stalled replication forks, an increased frequency of DNA breaks, and subsequent rearrangement by nonallelic homologous recombination or other similar mechanisms (20, 21). CNVs are highly homologous DNA sequences for which germline copy number varies between healthy individuals (22, 23). CNVs have been proposed to facilitate the generation of chromosomal alterations, similar to fragile sites (21, 24, 25).

We compared the number of observed breaks within 25 kB of a CNV to the frequency expected by chance alone on the basis of permuted data. In the Cisplatin-1 cohort, of 517 N_tAI breakpoints, 255 (49%) were associated with overlapping CNVs. Similarly, in the cisplatin-2 cohort, of 599 N_tAI breakpoints, 340 (57%) were associated with CNVs. In both trials, the observed number of N_tAI breaks associated with CNVs was significantly greater than expected by chance (Fig. 4A and B). Thus, many of the breakpoints leading to telomeric AI in TNBC occur near CNVs, suggesting that stalled replication forks, replication stress, or other CNV-associated mechanisms may be involved in the genesis of telomeric AI.

In our previous report of the Cisplatin-1 trial, we found an association between low BRCA1 transcript levels and better response to cisplatin (11). In the more recent trial, Cisplatin-2, BRCA1 transcript levels measured by quantitative real-time PCR (qRT-PCR) are also associated with cisplatin response (P = 0.015; Fig. 5A). In a combined analysis of data from both trials, lower BRCA1 transcript levels are associated with methylation of the BRCA1 promoter (P = 0.027; Fig. 5B), although BRCA1 promoter methylation itself is not significantly associated with cisplatin response (P = 0.25; Fisher exact test). BRCA1 mRNA levels are inversely associated with N_tAI in the 2 cisplatin trials (r_s = −0.50, P = 0.0053; Fig. 5C). This finding suggests that dysfunction of a BRCA1-dependent process or other abnormality causing low BRCA1 mRNA may be responsible for the high level of telomeric AI and also cisplatin sensitivity in many of these TNBCs.

ROC analysis of the combined TNBC trials suggests that BRCA1 expression level or N_tAI may provide a similar predictive accuracy for cisplatin sensitivity (Supplementary Fig. S6A). When high N_tAI and low BRCA1 expression are combined in a predictive model, the positive predictive value and specificity of prediction improved considerably, but the sensitivity was decreased relative to N_tAI alone (Supplementary Fig. S6B), suggesting that low BRCA1 expression does not account for all cisplatin-sensitive tumors.

In the TNBC trials, we noted a few cisplatin-sensitive tumors with high levels of N_tAI but high BRCA1 mRNA, suggesting that alternative mechanisms may drive the generation of tAI in some tumors. Analysis of TCGA data of ER⁻/HER2⁻ breast cancer and wtBRCA serous ovarian cancer reveals an inverse correlation between N_tAI and BRCA1 expression. Yet, in both cohorts, there was a considerable subset of tumors with high N_tAI and high BRCA1 expression (Supplementary Fig. S7A and B). Unlike N_tAI, BRCA1 expression was not apparently different between sensitive and resistant wtBRCA serous ovarian cancers (Supplementary Fig. S7C). These findings suggest a model whereby high N_tAI may represent a readout of DNA repair deficiency resulting from either low BRCA1 expression or from other known or unknown mechanisms (Fig. 6).

Our study analyzed 2 preoperative clinical trials in women with TNBC treated with cisplatin, in which pathologic response at the time of surgery provided an experimental endpoint. Sporadic TNBCs are heterogeneous in their responses to platinum salts, which are chemotherapeutic agents that depend in part on DNA repair defects for their cytotoxic activity (26, 27). Lesions in DNA repair caused by BRCA1 or BRCA2 dysfunction lead to platinum sensitivity; we reasoned that the types of chromosomal aberrations arising in the context of BRCA dysfunction might also be associated with platinum sensitivity in wtBRCA cancers.

On the basis of results in cell lines, we chose to enumerate one such chromosomal abnormality, telomeric AI, in pretreatment tumor genomes and to relate this to pathologic response after cisplatin therapy. N_tAI was associated with response to platinum treatment in our TNBC cisplatin trials and in platinum-treated serous ovarian cancer, and these findings suggest that the burden of this genomic abnormality exposes an underlying deficiency of DNA repair in the platinum-sensitive subset of these cancers. AI propagated from a given chromosomal location to the telomere suggests the operation of error-prone processes giving rise to abnormal cross-over or template switching events, rather than error-free DNA repair.

We found that the breakpoints of telomeric AI regions are nonrandom and enriched for CNVs. This pattern also suggests defective DNA repair. CNVs are associated with other repeat sequences, such as Alu repeats, are concentrated in pericentromeric and subtelomeric regions, and are associated also with common fragile sites (28, 29). These repeat elements are thought to result in replication “slow zones,” which are prone to replication stalling and formation of DNA double-strand breaks (30, 31). Furthermore, downregulation of Rad51 or inhibition of BRCA1 increases the fragility at such sites when cells are under replication stress (32, 33). The observed association of low BRCA1 expression levels in many tumors with high N_tAI suggests deficient homologous recombination, impaired S- or G₂–M checkpoint function, or a combination of these factors underlying the generation of this type of genomic abnormality.

Cisplatin forms interstrand cross-links on DNA that lead to stalled replication forks and DNA double-stand breaks that must be repaired if the cell is to survive. It is likely that these breaks are repaired by the use of similar mechanisms to those used at stalled replication forks and DNA breaks generated at sites of CNVs. Therefore, high pretreatment N_tAI identifies tumors unable to accurately repair breaks and restart stalled replication forks at sites of CNV. These same tumors are also unable to contend with stalled forks at sites of cisplatin cross-links.

Although AI at sites of CNV may reflect inefficient error-free repair, other explanations should be considered. Both TNBC cohorts showed a significant relationship between N_tAI and pathologic response to cisplatin chemotherapy. Nevertheless, there were patients in both trials whose tumors showed poor response to cisplatin therapy despite having high N_tAI. Similarly, a few of the BRCA1-mutated ovarian cancers had high N_tAI yet were resistant to platinum therapy. Because N_tAI is a summation of ongoing and past DNA lesions, resistance mechanisms acquired after generation of tAI would confound the relationship between N_tAI and response. In carriers of BRCA1 or BRCA2 mutations, some tumors that become resistant to platinum agents carry a reversion mutation that partially or completely restores BRCA1 or BRCA2 function and restores homologous recombination (26, 27, 34).

Reversion has also been seen in a cell line with a BRCA2 mutation selected for PARP inhibitor resistance (27). Reversion mutations and in cis compensating mutations were observed in patients with Fanconi anemia, resulting in improvement in their bone marrow function (35). Inactivation of TP53BP1 restores the balance between homologous recombination and nonhomologous end joining in BRCA1-mutated cells and renders them resistant to PARP inhibitors (36, 37). Finally, drug transporters may prevent accumulation of platinum agents in tumor cells (38). Therefore, reversion of or compensation for a preexisting DNA repair defect may generate a tumor with high N_tAI but resistance to platinum treatment; other platinum resistance mechanisms unrelated to DNA repair would have the same effect.

Our analysis begins to suggest an outline of the molecular taxonomy of TNBC and ovarian cancer with respect to DNA repair and drug sensitivity. Most platinum-resistant breast or ovarian cancers are tumors with repair proficiency and low N_tAI. Two subsets of wtBRCA tumors possess high N_tAI and are sensitive to platinum-containing drugs. In one of these subsets, repair deficiency may be the consequence of low BRCA1 expression and, in the other subset, repair may be crippled by mechanisms that do not depend upon BRCA1 expression. These observations will no doubt be further refined; inclusion of reversion mutations, compensations by other events in DNA repair pathways, other mechanisms of drug resistance, and other as-yet unappreciated factors may help to enhance our prediction of drug sensitivity in the future.

In conclusion, a summary measure of telomeric chromosome aberrations in the tumor genome, N_tAI, predicts sensitivity to platinum treatment. Our findings implicate N_tAI as a marker of impaired DNA double-strand break repair. Assays to determine N_tAI are feasible via the use of formalin-fixed paraffin embedded tumor material, and recent algorithms such as ASCAT permit the accurate determination of copy number and AI in a majority of samples despite low tumor cell content. N_tAI may prove useful in predicting response to a variety of therapeutic strategies exploiting defective DNA repair.

Breast cancer cell lines were originally obtained from American Type Culture Collection and were most recently authenticated by Promega PowerPlex 1.2 short tandem repeat profiling at the DF/HCC microarray core laboratory in September 2011. Drug sensitivity measurements in breast cancer cell lines BT20, BT549, HCC1187, HCC1143, MDA-MB-231, MDA-MB-468, HCC38, MDA-MB-453 (triple-negative), CAMA-1, MCF7, T47D (ER⁺), BT474, HCC1954, and MDA-MB-361 (HER2⁺) were originally generated for a separate study in which it was reported as “data not shown” in a recently published article (13).

To summarize in brief, cells were exposed to a series of concentrations of various chemotherapeutic agents for 48 hours. Viable cell number was quantified by the use of CellTiter 96 AQueous One Solution Cell Proliferation Assay according to the manufacturer's instructions (Promega). Drug sensitivity was quantified as the dose of drug resulting in a 50% reduction of growth (IC₅₀). We found MCF7 to be highly resistant to all of the chemotherapeutic agents tested, consistent with its reported caspase-3 deficiency and resistance to drug induced apoptosis (39). In our analyses with measures of genomic aberration, MCF7 was the only clear outlier, and for these reasons, was excluded from our analyses.

For this study, subjects were included for analysis of response to cisplatin if they progressed on therapy or if they received at least 3 of 4 cycles of the planned cisplatin therapy, had received no other nonprotocol therapy before surgery, and if an adequate amount of tumor was available from the pretreatment biopsy. Therapeutic response was measured with the semiquantitative Miller–Payne grading system, which estimates the percentage of reduction in invasive tumor volume and cellularity on the basis of pathologic assessment of surgical samples after therapy (40). The Cisplatin-1 trial consisted of 28 mainly sporadic TNBC patients treated with preoperative cisplatin monotherapy, of whom 4 progressed on therapy and 24 completed 4 cycles of cisplatin therapy (11). Cisplatin-2 consisted of 51 TNBC patients treated with preoperative cisplatin and bevacizumab, of which 1 patient progressed on therapy and 44 patients completed 4 cycles of cisplatin therapy before surgery (12). Two patients included in this study were taken to surgery after completing 3 cycles of cisplatin therapy because of the development of toxicity; in both cases there was no appreciable pathologic response in the excised tumor after 3 cycles of cisplatin.

For both trials, core biopsies of tumor were obtained before initiation of treatment. Adequate tumor for analysis was present for 27 of 28 subjects in Cisplatin-1 and 37 of 51 subjects in Cisplatin-2. Hematoxylin and eosin–stained tissue sections of pretreatment core-needle biopsies were examined microscopically; for all biopsies for which enrichment was deemed feasible, sections were manually microdissected with the use of an 18-gauge needle. DNA was extracted by proteinase K and RNase A digestions, phenol/chloroform extraction, and ethanol precipitation. Paired normal DNA from patients was obtained from peripheral blood lymphocytes for all cases in Cisplatin-1 and from 10 cases in Cisplatin-2.

Public SNP array data, expression data, and clinical annotation data were obtained for the TCGA ovarian (19) and breast cancer cohorts from the TCGA web site (41). BRCA1 and BRCA2 mutation status for the ovarian cancers was obtained from cBIO data portal (42). In the ovarian cohort, we identified 218 samples with SNP data that passed ASCAT (see the section entitled “Genotyping and Copy Number Analysis”), BRCA mutation status, and interpretable clinical annotations for treatment and outcomes indicating initial treatment with adjuvant platinum-based chemotherapy, predominantly the combination of carboplatin and docetaxel. We classified “treatment sensitive” patients as those annotated as having shown a partial or complete response to initial treatment and no progression or recurrence within 6 months of initial treatment (n = 187); the term “treatment resistant” was applied to those annotated as having stable or progressive disease on initial therapy or disease recurrence or progression within 6 months (n = 31). In the breast cohort, we identified 78 samples with matched gene expression and SNP data that passed ASCAT, which were classified as ER⁻/HER2⁻ on the basis of clustering of the ESR1 and ERBB2 transcripts (see Supplementary Methods).

DNA was sent to Affymetrix, Inc. for determination of genotypes via use of the molecular inversion probe-based genotyping system, OncoScan FFPE Express (43). The commercial assay, which determines genotype of 330,000 SNPs, was used for analysis of the Cisplatin-2 trial. An early version of the OncoScan assay, which genotypes 42,000 SNPs, was used for the Cisplatin-1 trial. Allele signal intensity and genotypes from the OncoScan genotyping assay were processed and provided to us by Affymetrix. The OncoScan SNP genotype data for the cisplatin therapy trials were submitted to the National Center for Biotechnology Information Gene Expression Omnibus database under accession number GSE28330. Public SNP array raw data for the breast cancer cell lines were obtained from the Sanger Institute Catalogue Of Somatic Mutations In Cancer website (44, 45), public SNP array data from an independent breast cancer cell line study conducted by Heiser and colleagues (18), and public SNP array data from the TCGA ovarian (19) and breast cancer cohorts were preprocessed by the AROMAv2 and CalMaTe algorithms (46) and, when a paired normal sample was available, TumorBoost (47).

Processed genotype data from OncoScan genotyping and public SNP array data were analyzed for allele-specific copy numbers and tumor cell content by the aforementioned algorithm, ASCAT (10). ASCAT is designed to correct for normal cell contamination and tumor cell ploidy but occasionally fails to fit a model to a given sample. In this study, ASCAT failed to process 3 of 14 cell lines from Sanger, 15 of 42 cell lines from Heiser and colleagues (18), and 5 of 37 samples from the Cisplatin-2 trial. AI was defined as any time the copy number of the 2 alleles were not equal, and at least one allele was present (Supplementary Fig. S1). To ensure that all trial cases were comparable, we eliminated cases estimated by ASCAT to have less than 36% tumor cell content, the highest level of normal cell admixture in the Cisplatin-1 trial, which was the trial with an overall greater tumor purity. Thus, we included all 27 samples with SNP array data from the Cisplatin-1 trial and 26 of 32 samples with SNP array data that passed ASCAT from the Cisplatin-2 trial.

A minimum number of consecutive probes showing an aberration was required to call regions of AI and copy number change with confidence. To ensure similar aberration detection across the 3 platforms that were used, the minimum number of probes required to define a region of aberration was set to be proportional to the overall SNP density of the platform. The probe densities of the platforms were 42,000/genome OncoScan (prototype), 330,000/genome OncoScan FFPE Express, and 900,000/genome SNP6.0 for an approximate ratio of 1:8:20. Minimum probe requirements of 25 probes for 42-k OncoScan prototype, 200 probes for 330-k OncoScan FFPE Express, and 500 probes for SNP6.0 platform were chosen on the basis of optimizing for correlation of aberration measurement in a subset of samples with replicate data generated on both versions of the OncoScan platform (see also Supplementary Methods).

Telomeric AI and telomeric copy number change were defined as regions that extend to one of the subtelomeres but do not cross the centromere. Copy number of telomeric AI regions was defined as the mean copy number of the probes mapping to the region. Copy loss was defined as a mean of less than 1.5 copies, and copy gain was defined as a mean of greater than 2.5 copies. Association between N_tAI and response to cisplatin was measured by the AUC of the ROC curve for binary response. Correlation was determined by the Spearman rank correlation coefficient. Statistical significance was assessed by the Wilcoxon rank sum test. All P values are 2-sided.

The genomic location of common CNVs was acquired from the Database of Genomic Variants (48). Mapping for HG17 and HG18 was acquired to match the SNP probe mapping of the 42-k prototype and 330-k commercial OncoScan platforms, respectively. CNVs were considered associated with a breakpoint if they overlapped within a 25-kb window on either side of the breakpoint. To test for enrichment, we performed 1,000 permutations for each cohort, where we randomly shuffled the location of the DNA breakpoints based on the location of the SNP probes, and determined how many were associated with CNVs.

BRCA1 exon 16/17 and RPLP0 (control) qRT-PCR assay was performed as previously described (11) by the use of amplified tumor cDNA generated with the Ovation RNA Amplification System V2 kit (NuGen Technologies, Inc.). BRCA1 promoter methylation assay was performed as previously described (11). To combine the qRT-PCR transcript data from the Cisplatin-1 and Cisplatin-2 trials, the data were z-transformed within each cohort by centering it and dividing by the standard deviation.

Public normalized and summarized Agilent-based gene expression data were acquired from the TCGA for all breast cancer samples (level 3). Raw Affymetrix CEL files were obtained for ovarian cancer samples (level 1). Expression data for all TCGA ovarian cancer samples were normalized and summarized by the use of robust multiarray average, and the probe set “204531_s_at” was identified as the optimum probe set for measuring BRCA1 expression with the R package “JetSet” (49).

A.L. Richardson, Z. Szallasi, Z.C. Wang, D.P. Silver, A.C. Eklund, and N.J. Birkbak are inventors on a pending patent application based on this work.

The authors thank Affymetrix scientists Malek Faham, Yuker Wang, and Victoria Carlson, who performed the tumor genotype analysis of Cisplatin-1 by using an early version of OncoScan FFPE Express under a research agreement with Affymetrix, Inc.

This work is partly supported by the Dana-Farber/Harvard SPORE in Breast Cancer, grant number CA089393, by an Avon supplement to 5 P30 CA06516 Dana-Farber/Harvard Cancer Center Support Grant, and by the V Foundation for Cancer Research. J.E. Garber, N. Tung, Z.C. Wang. J.D. Iglehart, Z. Szallasi, and A.L. Richardson are supported by the Breast Cancer Research Foundation. N.J. Birkbak, A.C. Eklund, Q. Li, and Z. Szallasi are supported by The Danish Council for Independent Research Medical Sciences (FSS). D.P. Silver gratefully acknowledges support of the Cogan Family Foundation.