Background
Germline mutation in the
BRCA1 gene is associated with an increased lifetime risk of breast cancer alongside earlier disease onset and predisposition to the more aggressive triple-negative disease subtype [
1‐
4]. The enhanced risk and high penetrance of breast cancer due to a
BRCA1 germline mutation are attributable to the tumor-suppressor role of the BRCA1 protein, which modulates homologous recombination (HR)-dependent DNA repair [
4‐
6].
BRCA1-related HR deficiency is associated with large-scale chromosomal breaks, extensive copy number alterations, and genome instability [
7,
8]. However, HR deficiency is not limited to cancers carrying a
BRCA1 mutation. Epigenetic inactivation of
BRCA1, as well as germline or somatic alteration of other HR-family genes, can serve as alternative mechanisms driving HR deficiency, resulting in a BRCA1-like phenotype also known as BRCAness [
3,
4,
9‐
11]. Similar to
BRCA1-mutated cancers, BRCA1-like cancers are aggressive and typically exhibit poor prognosis. However, BRCA1-like cancers are more sensitive to chemotherapy, evidenced in both experimental work and patient studies [
4,
10‐
12]. Lacking HR DNA repair can selectively sensitize BRCA1-deficient cancer cells to DNA cross-linking, alkylating, and double stranded break-inducing agents as well as poly-ADP ribose polymerase (PARP) inhibitors [
5,
6], and improved survival outcomes are observed after high-dose platinum-based chemotherapeutic and PARP inhibitor treatment in patients with BRCA1-like breast tumors [
10,
11,
13]. The TALORx trial evaluating the potential benefit of chemo-endocrine versus endocrine therapy alone in patients with hormone-receptor-positive human epidermal growth factor 2 receptor (HER2)-negative cancer and intermediate OncotypeDX, recurrence scores showed mostly equivocal results [
14]. However, some benefit of chemotherapy was observed in younger women with intermediate scores, potentially attributable to responses in patients with hormone-receptor-positive BRCA1-like tumors, which are diagnosed at a significantly younger age than non-BRCA1-like tumors.
BRCA1-like breast tumors harbor extensive, characteristic genomic alterations. Genomic analyses show the distinct molecular patterning of
BRCA1-mutated, HR-deficient cancers compared to BRCA2-mutated amd HR-proficient cancers [
15‐
17]. Another pronounced feature of BRCA1-like, HR-deficient cancers is the extensive copy number alterations. This molecular hallmark motivated the classification of HR-deficient tumors based on their copy number profiles. Initially, array comparative genomic hybridization (aCGH) copy number was used to characterize the BRCA1-like phenotype and led to the development of a tool to predict breast cancer in patients with a
BRCA1 mutation or promoter hypermethylation [
10,
11,
18]. The aCGH copy-number features that distinguish BRCA1-like tumors led to the development of the BRCA1ness-MLPA assay, an experimental gold standard currently being tested in the clinical setting [
19,
20]. More recently, the classification of HR deficiency has been adapted to measurement of copy number using higher-resolution approaches [
11,
21].
A few studies have begun to characterize the molecular differences associated with BRCA1-related HR deficiency. HR-deficient cancers tend to exhibit more severe mutational burden and distinct mutational signatures [
3,
15,
22]. Transcriptome-wide alterations have also been reported and used for defining HR-deficient gene signatures [
12,
23,
24]. Further, HR deficiency is associated with global epigenetic changes and aberrant methylation of several HR family genes in cultured cells [
25,
26]. However, these initial assessments of BRCA1-like molecular or cellular profiles often had limited sample sizes and varying results. Moreover, a description of biological differences between BRCA1-like and non-BRCA1-like tumors in large-scale cancer cohorts is currently lacking. Further, while prior work has shown the highly dysregulated epigenetic landscape in breast tumors compared to the normal breast, especially at early stages of cancer [
2,
27], little is known about the epigenetic patterning of HR-deficient, BRCA1-like breast tumors relative to their non-BRCA1-like counterpart.
Here, we retrained and evaluated a classifier to identify BRCA1-like tumors using genome-wide copy number profiles, which can be measured by multiple platforms including genotyping array, methylation array, and next-generation sequencing [
21]. We then applied this classifier to identify tumors exhibiting the HR-deficient, BRCA1-like phenotype in two large-scale breast cancer cohorts: The Cancer Genome Atlas (TCGA) [
2] and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohorts [
28,
29]. In TCGA, for example, we detected nearly one third of breast tumors ofh the BRCA1-like phenotype, while only approximately 3% tumors had a
BRCA1 somatic alteration. Subsequently, we compared molecular, clinical, and epigenetic characteristics associated with the BRCA1-like phenotype, restricting our analyses to hormone-receptor-positive breast cancer (i.e. breast tumors expressing estrogen receptor (ER), progesterone receptor (PR), and/or human epidermal growth factor receptor 2 (HER2). We focused on these tumors because we anticipate their distinct molecular profile, which could render them responsive to the cytotoxic chemotherapy typically given to patients with triple negative breast cancer (TNBC) [
30‐
32].
Discussion
In this study, we retrained a BRCA1-like classifier using genome-wide copy number and validated the classifier by
in silico and experimental approaches. We estimated that 22% of all TCGA and METABRIC breast tumors were BRCA1-like, consistent with existing literature [
4,
15]. Notably, 13% hormone-receptor-positive tumors were BRCA1-like. Therapeutic strategies such as cytotoxic chemotherapy more commonly used in the triple-negative disease setting might be an effective alternative for treating these tumors.
Among hormone-receptor-positive breast tumors, the BRCA1-like phenotype is associated with increased mutational burden, as demonstrated by elevated mutation rates. Expression of Ki-67, a surrogate marker for cellular proliferation, was increased in BRCA1-like receptor-positive tumors. These molecular hallmarks serve as evidence supporting the more aggressive character of BRCA1-like tumors.
The genome-scale DNA methylation profile of BRCA1-like tumors, identified by our SVM classifier, appeared distinct. Furthermore, we detected hypermethylation of gene sets related to chromatin and nucleosome assembly. Of note, the
BRCA1 locus showed increased DNA methylation in BRCA1-like tumors, supporting the existing concept that hypermethylation of HR-family genes could serve as a driver for HR deficiency. Detecting hypermethylation and reduced gene expression of miR124–2, a negative regulator of cell proliferation [
52], is consistent with our Ki-67 gene expression analysis and further supports BRCA1-like tumors having a more aggressive phenotype.
Subsequently, differential methylation analysis comparing SVM-predicted BRCA1-like and non-BRCA1-like tumors identified 202 hypomethylated and 148 hypermethylated CpGs. Unsupervised hierarchical clustering of all 350 CpGs revealed a distinct “BRCA1-like cluster”, implying the potential utility of genome-scale DNA methylation as another biomarker to identify HR-deficient cancers, possibly in breast cancer biopsies shown to have similar methylation profiles to larger surgical blocks [
55]. We also noticed the increased heterogeneity in this cluster relative to the “non-BRCA1-like cluster”, a hallmark of aggressive cancers [
56]. In addition, this finding parallels the prior observation that when compared to normal-adjacent breast tissue, breast tumors exhibit increased heterogeneity [
2]. Moreover, our precise identification of differentially methylated CpGs, genes and gene sets allows focused investigation in the future, thereby enabling the identification of effective pharmacologic and therapeutic strategies in the future.
We observed members of the
HOX gene cluster to be hypermethylated. In line with this, many developmentally related pathways were found to be mildly enriched though not statistically significantly. These findings indicate that development and differentiation-related signaling pathways are characteristic of the HR-deficient, BRCA1-like phenotype. We followed up with this postulate by comparing DNA methylation age - a metric inferred from genome-scale DNA methylation profiles and related to cellular differentiation potential [
46] – between BRCA1-like and non-BRCA1-like tumors. In line with our differential methylation and pathway analysis, DNA methylation age was significantly lower in BRCA1-like tumors indicating a more poorly differentiated tumor state. These observations were overall consistent with prior works demonstrating that tumors with
BRCA1/2-related HR deficiency tend to be poorly differentiated or undifferentiated [
57].
Recent studies have shown that
BRCA1-deficient and
BRCA2-deficient genomes, despite both having HR loss, may nevertheless differ [
15‐
17]. Therefore, to better understand HR deficiency and chemotherapeutic sensitivity, development and characterization of molecular signatures that more broadly characterize the HR-deficient phenotype may be necessary.
One challenge was identifying HR-deficient, BRCA1-like tumors using a strict probabilistic threshold. Here, we used the cutoff of 0.50, which could be rather conservative. Despite having used a robust cross validation-based machine learning approach, there will be opportunities in the future to potentially improve the performance of our SVM classifier, with better balance among breast cancer subtypes in the training data. We acknowledge the limitation of cell lines in the experimental validation set, and note that future studies would benefit from inclusion of larger human sample sets for validation. Biologically, as seen in the TALORx trial where younger patients (age < 50 years) had improved chemotherapy response [
14], we also suspected that confounders such as patient age strata could influence the performance of BRCA1-like classifiers and the molecular characteristics of these tumors. While our Kaplan-Meier analysis showed some evidence that ER/PR-positive, HER2-negative breast cancer with the BRCA1-like phenotype was associated with worse overall survival, our results were not statistically significant in a covariate-adjusted Cox regression model. A possible explanation is the heterogeneity of the treatment regimens among study participants. We therefore anticipate that the application of our SVM BRCA1-like classifier to cohorts with more consistent treatment will have greater clinical value.
Acknowledgements
We acknowledge the U.S. National Institute of Health for funding support (R01DE022772, R01CA216265, and P20GM104416/6369 to BCC, KL2TR001088 to CC, and P20GM113132 to ANK), the Norris Cotton Cancer Center Prouty Pilot Funding to ANK and TWM, and the Burroughs-Wellcome Big Data in the Life Sciences Fellowship to YC. We thank the Dartmouth Molecular Biology Shared Resources and Research Computing for assistance.