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. 2010 Dec 15;16(24):6159-68.
doi: 10.1158/1078-0432.CCR-10-1027. Epub 2010 Aug 27.

A marker of homologous recombination predicts pathologic complete response to neoadjuvant chemotherapy in primary breast cancer

Monika Graeser  1 , Afshan McCarthy, Christopher J Lord, Kay Savage, Margaret Hills, Janine Salter, Nicholas Orr, Marina Parton, Ian E Smith, Jorge S Reis-Filho, Mitch Dowsett, Alan Ashworth, Nicholas C Turner
Affiliations

A marker of homologous recombination predicts pathologic complete response to neoadjuvant chemotherapy in primary breast cancer

Monika Graeser et al. Clin Cancer Res. .

Abstract

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 = 0.031) and high baseline Ki67 (P = 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 = 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 = 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.

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Figures

Figure 1
Figure 1. Development of RAD51 focus assay
A. HR competent PIR (Left) and HR incompetent BRCA2 mutant CAPAN1 cells (Right) were irradiated in vitro with 10Gy, and 6 hours post irradiation examined by immunofluorescent confocal microscopy. Red – RAD51, Green - γH2AX, Blue – DAPI nuclear stain. B. and C. Xenografts of CAPAN1, PIR, or CAL51 breast cancer cells were irradiated with 6Gy, or not, and 8 hours post irradiation tumour excised and fixed in formalin. FFPE sections were examined by immunofluorescent confocal microscopy. Red – RAD51, Blue – DAPI nuclear stain. B. Representative images post irradiation. C. Quantification of percentage of tumour cells with RAD51 foci in sections from five xenografts per condition. Post irradiation raw RAD51 count CAPAN1 5.3%, PIR 25.3%, CAL51 30.0% (p<0.0001 CAL51 vs CAPAN1 and p<0.0001 PIR vs CAPAN1, Student's T test).
Figure 2
Figure 2. RAD51 and geminin immunofluorescence in core biopsies of primary breast cancer
Confocal images of an HR competent and deficient primary breast cancers, with immunofluorescence staining of RAD51 (red) with DAPI counter stain (Blue) (Top panels), RAD51 and geminin (green) overlay with DAPI counter stain (Middle panels), and RAD51 - geminin overlay (bottom panels). A. Images from baseline biopsy (left) and 24 hrs post chemotherapy (right) from a tumour demonstrating induction of RAD51 foci in geminin positive cells following chemotherapy. B. Images from baseline biopsy (left) and 24 hrs post chemotherapy (right) from a tumour demonstrating no induction of RAD51 foci despite high geminin expression.
Figure 3
Figure 3. Raw RAD51 count shows a biphasic relationship with proliferation in 24 hour post chemotherapy biopsies
A. Plot of raw RAD51 counts (x axis, percentage of tumour cells that have RAD51 foci) against proliferative fraction assessed on same section (y axis, percentage of tumour cells with nuclear geminin expression) in core biopsies from 57 tumours taken at 24 hours post chemotherapy. In tumours with a raw RAD51 count >2% there is a positive correlation between RAD51 count and proliferation (ρ=0.67, 95% CI 0.45-0.82, p<0.0001, Spearman's correlation coefficient), but not in tumours with raw RAD51 count <2% (r=−0.21, p=NS). Open circles indicate tumours subsequently assessed as having a low RAD51 score (Figure 4). B. Assessment of the relationship between geminin positive and RAD51 positive cells. For each tumour the percentage of geminin positive cells that had RAD51 foci, and the percentage geminin negative cells that had RAD51 foci was assessed. Displayed are the results from all 57 tumours, with the percentage of geminin positive cancer cells that have RAD51 foci (median 40%, left), versus the percentage of geminin negative cancer cells that have RAD51 foci (median 0%, right). RAD51 foci are almost exclusively expressed in geminin positive cells (p<0.0001 Mann-Whitney U Test).
Figure 4
Figure 4. Assessment of RAD51 score in primary breast cancer and relationship with pathological complete response to chemotherapy and tumour subtypes
A. RAD51 score (Percentage of geminin postive cells that have RAD51 foci) in biopsies taken at 24 hours post chemotherapy from 57 primary breast cancers, displayed in order RAD51 score. Tumours with 100% of geminin positive cells having RAD51 foci are displayed on the left, and tumours were no RAD51 foci in geminin positive cells on the right. A tumour was defined as being HR defective with a RAD51 score <10% (indicated with a vertical dotted line). RAD51 score was assessed blinded to clinicopathological and chemotherapy response data. B. Data on pathological response to chemotherapy in post chemotherapy surgical specimen. Tumours that achieved a pathological complete response (pathCR) to neoadjuvant chemotherapy had lower RAD51 scores than those that did not achieve pathCR (Median RAD51 score; pathCR 2.6% vs non-pathCR 44%, p=0.028 Mann-Whitney U Test). Pathological response data was not available on 9 cases as discussed in Supplementary figure 1. C. Data on tumour subtype according to ER and HER2 status. Tumours negative for ER and HER2 (TN breast cancers) had lower low RAD51 scores than other breast cancer subtypes (Median RAD51 score; TN cancers 2.3% vs other subtypes 42.5%, p=0.0077 Mann-Whitney U Test).
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