Purpose: To assess the prevalence of defective homologous recombination (HR)-based DNA repair in sporadic primary breast cancers, examine the clincopathologic features that correlate with defective HR and the relationship with neoadjuvant chemotherapy response.Experimental Design: We examined a cohort of 68 patients with sporadic primary breast cancer who received neoadjuvant anthracylcine-based chemotherapy, with core biopsies taken 24 hours after the first cycle of chemotherapy. We assessed RAD51 focus formation, a marker of HR competence, by immunofluorescence in postchemotherapy biopsies along with geminin as a marker of proliferative cells. We assessed the RAD51 score as the proportion of proliferative cells with RAD51 foci.Results: A low RAD51 score was present in 26% of cases (15/57, 95% CI: 15%-40%). Low RAD51 score correlated with high histologic grade (P ¼ 0.031) and high baseline Ki67 (P ¼ 0.005). Low RAD51 score was more frequent in triple-negative breast cancers than in ER-and/or HER2-positive breast cancer (67% vs. 19% respectively; P ¼ 0.0036). Low RAD51 score was strongly predictive of pathologic complete response (pathCR) to chemotherapy, with 33% low RAD51 score cancers achieving pathCR compared with 3% of other cancers (P ¼ 0.011).Conclusions: Our results suggest that defective HR, as indicated by low RAD51 score, may be one of the factors that underlie sensitivity to anthracycline-based chemotherapy. Defective HR is frequent in triple-negative breast cancer, but it is also present in a subset of other subtypes, identifying breast cancers that may benefit from therapies that target defective HR such as PARP inhibitors. Clin Cancer Res; 16(24); 6159-68. Ó2010 AACR.DNA double-strand break repair by homologous recombination (HR) is required for the repair of DNA damage arising from many currently used DNA-damaging chemotherapy agents (1), as well as DNA single-strand breaks that are generated by inhibitors of poly(ADP ribose) polymerase 1 (PARP; ref. 2). The tumor suppressor genes BRCA1 and BRCA2 encode proteins that are required for HR (3), and tumors arising in carriers of germline mutations in BRCA1/BRCA2 are defective in HR and, consequently, are highly sensitive to DNA-damaging chemotherapy (4, 5) and PARP inhibitors (6). Increasing evidence suggests that a proportion of sporadic breast cancers may also have defects in HR (1). Sporadic basallike breast cancers have low BRCA1 expression (7-9) and BRCA1 promoter methylation is found in approximately 10% of sporadic breast cancers including nonbasal tumors (7). Recently, it has also been proposed that loss of PTEN expression may also lead to a defect in HR (3,10,11).In this study, we set out to develop a functional assay for HR with the purpose of assessing the prevalence of a functional defect in HR in sporadic breast cancer and examine whether defective HR correlated with clinicopathologic features and sensitivity to chemotherapy. The protein RAD51 is a DNA recombinase and key component of HR (12). In response to DNA double-strand bre...

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Purpose: Digital PCR is a highly accurate method of determining DNA concentration. We adapted digital PCR to determine the presence of oncogenic amplification through noninvasive analysis of circulating free plasma DNA and exemplify this approach by developing a plasma DNA digital PCR assay for HER2 copy number.Experimental Design: The reference gene for copy number assessment was assessed experimentally and bioinformatically. Chromosome 17 pericentromeric probes were shown to be suboptimal, and EFTUD2 at chromosome position 17q21.31 was selected for analysis. Digital PCR assay parameters were determined on plasma samples from a development cohort of 65 patients and assessed in an independent validation cohort of plasma samples from 58 patients with metastatic breast cancer. The sequential probability ratio test was used to assign the plasma DNA digital PCR test as being HER2-positive or -negative in the validation cohort.Results: In the development cohort, the HER2:EFTUD2 plasma DNA copy number ratio had a receiver operator area under the curve (AUC) ¼ 0.92 [95% confidence interval (CI), 0.86-0.99, P ¼ 0.0003]. In the independent validation cohort, 64% (7 of 11) of patients with HER2-amplified cancers were classified as plasma digital PCR HER2-positive and 94% (44 of 47) of patients with HER2-nonamplified cancers were classified as digital PCR HER2-negative, with a positive and negative predictive value of 70% and 92%, respectively.Conclusion: Analysis of plasma DNA with digital PCR has the potential to screen for the acquisition of HER2 amplification in metastatic breast cancer. This approach could potentially be adapted to the analysis of any locus amplified in cancer.