A mutational signature reveals alterations underlying deficient homologous recombination repair in breast cancer

Abstract

Biallelic inactivation of BRCA1 or BRCA2 is associated with a pattern of genome-wide mutations known as signature 3. By analyzing ∼1,000 breast cancer samples, we confirmed this association and established that germline nonsense and frameshift variants in PALB2, but not in ATM or CHEK2, can also give rise to the same signature. We were able to accurately classify missense BRCA1 or BRCA2 variants known to impair homologous recombination (HR) on the basis of this signature. Finally, we show that epigenetic silencing of RAD51C and BRCA1 by promoter methylation is strongly associated with signature 3 and, in our data set, was highly enriched in basal-like breast cancers in young individuals of African descent.

Figure 1:. Characterization of four distinct mutational signatures in breast cancer.

A Bayesian non-negative matrix factorization approach (Methods) was employed to decompose the overall mutational spectrum across a cohort of 992 breast cancer samples into distinct mutational signatures (“SNVs” – top row). Each color represents one of the six potential base substitutions, with each substitution further stratified by the adjacent 5’ and 3’ flanking nucleotides. The pattern of each signature was matched against a database of known signatures and their etiologies (Signature 3, C>T_CpG, APOBEC, MSI). A signature characterized by C>G transversions and C>T transitions at TC[A/T] motifs (in which the altered cytosine is flanked by a 5’ thymine and a 3’ adenine or thymine) is likely attributable to mutagenesis via endogenous APOBEC machinery (“APOBEC”; COSMIC signatures 2 and 13). A second signature primarily consisting of C>T transitions at CpG dinucleotides has been described among all tumor types and is thought to arise from spontaneous 5-methyl-cytosine deamination (“C>T CpG”; COSMIC signature 1). The third signature resembles previously characterized signatures identified in samples known to have microsatellite instability (MSI) (“MSI”; COSMIC signature 6). A recurrent signature in the cohort closely approximates Signature 3, and is characterized by mutations in all 96 nucleotide contexts, with an increased rate among C:G basepairs as compared to A:T ( “Signature 3”).

Figure 2:. Overall mutation rates, mutational signature contributions, and clinicopathologic features per patient as sorted by descending Signature-3 activity.

Each column represents a tumor sample. Overall indel burden is shown in the top panel with insertion or deletion designated by color. The second panel demonstrates absolute mutation burden per sample, with the estimated contribution of each of the four mutational signatures designated by color (samples are arranged in descending order of Signature 3 activity). The third panel shows sample-level annotations with regard to lesions in known homologous recombination (HR) pathway genes, along with PAM50 intrinsic subtype data, and triple negative status by immunohistochemistry. The bottom panel exhibits genetic lesions by type in select HR genes with mutation type indicated by color. Shown are the first 250 samples by Signature 3 activity; see Supplementary Figure 3 for the rest 742 samples.

Figure 3:. Signature 3 activity in tumors with somatic, germline and epigenetic alterations in HR-pathway genes.

(A) Signature 3 activity is elevated in tumors with somatic, germline and epigenetic alterations in BRCA1/2. Tumors were stratified by germline, somatic or epigenetic events in BRCA1/2 and plotted by number of mutations attributable to Signature 3. Pathogenic germline events included those described by ClinVar, in addition to frameshift and nonsense variants. The median signature count among each group was tested against samples lacking any discernible BRCA1/2 alterations (denoted as “no BRCA event”) with two-sided Wilcoxon rank-sum test results as indicated. Open circles represent samples with mono-allelic lesions (i.e. without evidence of biallelic inactivation). Samples with epigenetic silencing are indicated by diamonds. Two samples which harbored a somatic and a germline truncating event in BRCA1/2 and are included in both somatic and germline categories (B) Signature 3 activity in patients with germline alterations as previously denoted. (C) Signature 3 activity is associated with somatic, germline and epigenetic alterations in HR components beyond BRCA1/2. Signature 3 activity was compared between samples lacking any of the noted alterations (“no event”) versus those with any genetic or epigenetic event in the HR pathway. Samples with an event in BRCA1/2 were noted by an asterisk. Comparison of Signature 3 activity was conducted with two-sided Wilcoxon rank-sum results noted by group

Figure 4:. Association of promoter methylation of RAD51C with elevated Signature 3 in basal-like tumors.

(A) Schematic representation of RAD51C architecture and its known interacting proteins that promote HR. (B) Scatter plot of RAD51C expression as compared to promoter methylation levels (methylation values higher than 0.2 were considered for epigenetic silencing). Samples with high Signature 3 activity (top quartile) are denoted by triangles (C) Elevated Signature 3 activity is associated with alterations among multiple components of the HR-pathway and a diversity of alteration types. Samples were ranked by the number of Signature 3 associated mutations and plotted in descending order in bins of 50. The top panels show alterations among various HR-pathway genes, while the bottom panel shows the types of alterations in these genes, demonstrating enrichment of events among the samples with the highest Signature 3 activity. (D) Signature 3 activity was significantly associated with RAD51C promoter methylation among basal-like breast cancers, but not among other intrinsic subtypes.

Figure 5:. Analysis of genetic and epigenetic events in the 250 samples with highest Signature 3 levels, broken by largest racial subgroups in our cohort (white and African American).

(A) Distribution of various types of alterations among patients stratified by racial groups show an enrichment of promoter methylation in African American patients. The inset shows the distribution of various types of alterations in individual genes. The enrichment of promoter methylation among African American patients is most pronounced in women aged 40–49, both when looking at all breast cancer subtypes (B) and only basal-like cancers (C).

Figure 6:. A framework for enhancing the classification of BRCA1/2 germline missense variants using signature 3 and biallelic inactivation.

Beginning with all rare BRCA1/2 missense germline variants (n=137; 3.5% of randomly selected variants in TCGA breast cancer dataset are pathogenic), the group was dichotomized by presence of LoH at the BRCA1/2 locus. Those without LOH (n = 82) included only one sample with a pathogenic allele (i.e. 1.2% probability of pathogenicity), although this sample also harbored an additional variant with the potential for biallelic inactivation (indicated by red frame). Conversely, among those with LOH (n= 60; 6.6% of variants are pathogenic), samples were further sorted by allelic imbalance (22.2% of variants are pathogenic), elevated Signature 3 activity (66.6% probability of pathogenicity, indicated by green frame) and the absence of lesions among other HR-pathway components

Figure 7:. Prediction accuracy of Signature 3 relative to established rearrangement-based HRD scores

(A) The three panels exhibit receiver operating characteristic (ROC) curves for predicted alterations among BRCA1, BRCA2, RAD51C, and PALB2 using Signature 3 levels. The panels show analyses based on (i) all pathogenic germline mutations (ii) biallelic pathogenic germline mutations and (iii) pathogenic germline, loss of function somatic, or epigenetic alterations. (B) ROC curves comparing Signature 3 performance to LOH and LST scores for identifying samples with the HR-associated lesions denoted above. (C) Scatter plot exhibiting a comparison of Signature 3 levels versus HRD-LOH (top panel) and HRD-LST scores (bottom panel).