Bi-allelic alterations in DNA repair genes underpin homologous recombination DNA repair defects in breast cancer

Abstract

Homologous recombination (HR) DNA repair-deficient (HRD) breast cancers have been shown to be sensitive to DNA repair targeted therapies. Burgeoning evidence suggests that sporadic breast cancers, lacking germline BRCA1/BRCA2 mutations, may also be HRD. We developed a functional ex vivo RAD51-based test to identify HRD primary breast cancers. An integrated approach examining methylation, gene expression, and whole-exome sequencing was employed to ascertain the aetiology of HRD. Functional HRD breast cancers displayed genomic features of lack of competent HR, including large-scale state transitions and specific mutational signatures. Somatic and/or germline genetic alterations resulting in bi-allelic loss-of-function of HR genes underpinned functional HRD in 89% of cases, and were observed in only one of the 15 HR-proficient samples tested. These findings indicate the importance of a comprehensive genetic assessment of bi-allelic alterations in the HR pathway to deliver a precision medicine-based approach to select patients for therapies targeting tumour-specific DNA repair defects. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: BRCAness; DNA repair; RAD51; homologous recombination-deficient; mutation.

Figure 1. Schematic of study design

Tumors were prospectively collected from 56 patients for ex-vivo functional assessment of the status of the HR pathway, using RAD51 foci analysis. Tumors were classified as HR deficient or proficient using this assay. A multi-faceted genomics approach, integrating whole-exome sequencing, analysis of germ-line mutations, copy number variation, gene expression, and methylation was then used to determine the underlying etiology of HRD.

Figure 2. RAD51, γH2AX, and BRCA1 nuclear foci analysis of representative RAD51-proficient and RAD51-deficient case and distribution of RAD51-deficiency in breast cancer

a.) RAD51, γH2AX, and BRCA1 foci in a homologous recombination HR-proficient breast cancer in mock-treated (left) and irradiated conditions (right). b.) Radiation-induced RAD51, γH2AX, and BRCA1 foci in a breast tumor with deficient HR in mock-treated (left) and irradiated conditions (right). c.) Quantification of RAD51, γH2AX, and BRCA1 foci in cells (n=200) from a tumor with proficient HR. Note strong increases in the number of cells with RAD51, γH2AX, and BRCA1 following 10 Gy of ionizing radiation (IR) (error bars indicate s.e.) d.) Quantification of foci in in cells (n=200) from a tumor with deficient HR. Note strong induction in γH2AX with IR, without an increase in RAD51 or BRCA1 foci. All statistical comparisons were performed by comparing two proportions with a Z-test. e.) Relative fold induction of RAD51 foci formation in the irradiated, compared with the un-irradiated condition for all tumors. The relative fold induction is calculated as the number of nuclei with > 5 foci in the irradiated state divided by the number of nuclei in the un-irradiated state. A bi-modal distribution in relative fold induction is demonstrated, with 11 tumors (black) exhibiting <1.25 fold induction of RAD51 foci and classified as functional HRD. f.) Distribution of RAD51-deficient tumors according to the clinical subtypes of breast cancers. Although RAD51-deficiency was numerically more frequent in triple-negative breast cancers, this was not statistically significant (TNBC, 42%, p=0.13, Fisher’s exact test). ER, estrogen receptor; pos, positive; neg, negative.

Figure 3. Association of Genomic ‘Scars’ with RAD51 status

a.) RAD51-deficient breast cancers harbor a higher LST score than RAD51-proficient cases (p=0.002). b.) ntAI scores by RAD51 status in RAD51-proficient and RAD51-deficient breast cancers (p=0.009). c.) RAD51-deficient breast cancers have a higher Myriad LOH/HRD score than RAD51-proficient cancers (p=0.048). d.) Breast tumors with an alteration in an HR Gene (Truncating/frame-shift mutation, homozygous deletion, or non-synonymous mutation with loss-of-heterozygosity) show significantly higher LST scores than those without a genetic alteration in an HR gene (p = 5.2*10⁻⁴). Wt, wild-type. All comparisons were performed using Wilcoxon rank-sum tests.

Figure 4. Relationship between RAD51 status and gene expression and methylation

a.) Normalized NanoString expression counts of homologous recombination (HR) DNA repair-related genes compared between DNA repair-deficient (HRD) and DNA repair-proficient tumors as determined by RAD51 foci formation. No individual gene expression was associated with RAD51 status (statistical comparisons performed with t-tests). Supervised hierarchical clustering was unrevealing. Bisulfite sequencing of BRCA1 promoter using primer sets for un-methylated and methylated PCR is indicated in annotation panel below RAD51 status. Note, data in figure is only shown for samples with both gene expression and methylation available, however statistical tests were performed with all available data. b.) Bisulfite sequencing of BRCA1 promoter using primer sets for unmethylated and methylated PCR. The presence of a product in the methylated reaction indicates the presence of methylation in BRCA1 promoter.

Figure 5. Genetic changes in HR genes in RAD51-deficient and proficient samples

The repertoire of large-scale state transitions (LSTs), the number of somatic insertions and deletions (indels), association with BRCA mutational signature, as well as germline and somatic genetic alterations in genes associated with homologous recombination are presented. Cases are ordered first by RAD51 status, then by increasing LST. The number of indels for each case is divided by size according to the color key. Cases with a BRCA-associated mutation signature are annotated (see Online Methods for details). The grid illustrates the germline and somatic genetic alterations in HR genes. The types of alterations are indicated in the color key on the right. PIK3CA and TP53 mutation status, receptor and RAD51 status, are annotated in the phenobar (top). Exon duplication refers to a duplication of exon 3 in the BRCA2 gene. ER, estrogen receptor; TNBC, triple-negative breast cancer.