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. 2009 Aug;7(8):1304-9.
doi: 10.1158/1541-7786.MCR-09-0149. Epub 2009 Aug 11.

Utility of DNA repair protein foci for the detection of putative BRCA1 pathway defects in breast cancer biopsies

Henning Willers  1 , Alphonse G Taghian, Chen-Mei Luo, Alejandro Treszezamsky, Dennis C Sgroi, Simon N Powell
Affiliations

Utility of DNA repair protein foci for the detection of putative BRCA1 pathway defects in breast cancer biopsies

Henning Willers et al. Mol Cancer Res. 2009 Aug.

Abstract

The DNA damage response pathway controlled by the breast cancer and Fanconi anemia (FA) genes can be disrupted by genetic or epigenetic mechanisms in breast cancer. Defects in this pathway may render the affected tumors hypersensitive to DNA-damaging agents. The identification of these defects poses a challenge because of the large number of genes involved in the FA/BRCA pathway. Many pathway components form subnuclear repair protein foci upon exposure to ionizing radiation in vitro, but it was unknown whether foci can be detected in live cancer tissues. Thus, the goal of this pilot study was to identify pathway defects by using a novel ex vivo foci biomarker assay on tumor biopsies. Fresh pretreatment biopsy specimens from patients with locally advanced sporadic breast cancer were irradiated or mock-treated in the laboratory (ex vivo). Foci formation of DNA repair proteins BRCA1, FANCD2, and RAD51 was detected by immunofluorescence microscopy. Three out of seven tumors showed intact radiation-induced foci formation, whereas the other four tumors exhibited a defective foci response. Notably, three of the foci-defective tumors were estrogen receptor/progesterone receptor/HER2-negative (triple-negative), a phenotype that has been associated with BRCA1 deficiency. In conclusion, in this pilot study, we report the successful detection of BRCA1, FANCD2, and RAD51 foci in breast cancer biopsies irradiated ex vivo. Our approach represents a potentially powerful biomarker assay for the detection of pre-existing and functionally important defects within the complex FA/BRCA pathway, which may ultimately allow us to tailor cancer treatment to the DNA repair profile of individual tumors.

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Figures

FIGURE 1
FIGURE 1
Detection of repair protein foci in fresh frozen breast cancer tissues. A. Illustration of the experimental approach. B. Representative images illustrating the detection of areas containing viable tumor as well as subnuclear RAD51, FANCD2, and BRCA1 foci.
FIGURE 2
FIGURE 2
A. Representative images illustrating the induction of RAD51 foci in three tumors (cases 56, 57, 59) following exposure of samples to ionizing radiation (IR) ex-vivo. B. Quantification of cells with RAD51 foci. Bars represent means with upper standard error. Relative fold induction with IR is given. P-value, unpaired t-test, two sided. C. RAD51 foci analysis analogous to panel B. Numbers indicate slide numbers per case.
FIGURE 3
FIGURE 3
A. Representative images as in Figure 2A. B. Quantitative analysis of foci as in Figure 2B
FIGURE 4
FIGURE 4
Identification of BRCA1-deficient tumors. A. Quantification of RAD51, FANCD2 and BRCA1 foci as in Figure 2B. Indicated is also the hormone receptor status for each of the four tumors. B. Quantification of RAD51 foci over several slides as in Figure 2C. C. Relative BRCA1 expression by quantitative RT-PCR is plotted against tumor classification based on foci analysis. Lines represent the logarithmic mean.
See this image and copyright information in PMC

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