Tumour lineage shapes BRCA-mediated phenotypes

Abstract

Mutations in BRCA1 and BRCA2 predispose individuals to certain cancers^1-3, and disease-specific screening and preventative strategies have reduced cancer mortality in affected patients^4,5. These classical tumour-suppressor genes have tumorigenic effects associated with somatic biallelic inactivation, although haploinsufficiency may also promote the formation and progression of tumours^6,7. Moreover, BRCA1/2-mutant tumours are often deficient in the repair of double-stranded DNA breaks by homologous recombination^8-13, and consequently exhibit increased therapeutic sensitivity to platinum-containing therapy and inhibitors of poly-(ADP-ribose)-polymerase (PARP)^14,15. However, the phenotypic and therapeutic relevance of mutations in BRCA1 or BRCA2 remains poorly defined in most cancer types. Here we show that in the 2.7% and 1.8% of patients with advanced-stage cancer and germline pathogenic or somatic loss-of-function alterations in BRCA1/2, respectively, selective pressure for biallelic inactivation, zygosity-dependent phenotype penetrance, and sensitivity to PARP inhibition were observed only in tumour types associated with increased heritable cancer risk in BRCA1/2 carriers (BRCA-associated cancer types). Conversely, among patients with non-BRCA-associated cancer types, most carriers of these BRCA1/2 mutation types had evidence for tumour pathogenesis that was independent of mutant BRCA1/2. Overall, mutant BRCA is an indispensable founding event for some tumours, but in a considerable proportion of other cancers, it appears to be biologically neutral-a difference predominantly conditioned by tumour lineage-with implications for disease pathogenesis, screening, design of clinical trials and therapeutic decision-making.

Extended Data Fig. 1:. Study cohort and BRCA1/2 germline and somatic mutation distribution.

a) The number of tumor and matched normal specimens are shown by cancer type. NSCLC, non-small cell lung cancer; CUP, cancer of unknown primary; GIST, gastrointestinal stromal tumor; SCLC, small-cell lung cancer; GINET, gastrointestinal neuroendocrine tumor; CNS, non-glioma central nervous system tumors; NHL, non-Hodgkin’s lymphoma. b) Somatic mutational burden (log₂ mutations per Mb) in tumors defined as non-hypermutated or hypermutated (via microsatellite instability, DNA polymerase epsilon mutations, or alkylating therapy-induced; see Methods). c) BRCA1 and BRCA2 somatic mutation rates in deciles of increasing tumor mutational burden. The highest mutational burden tumors also had the highest rate of BRCA1/2 mutations. d) The percent of tumors in each tumor type harboring either somatic VUS or LoF BRCA1/2 mutations as a function of the median somatic mutational burden of that cancer type (excluding hypermutated cases). Overall, the rate of somatic LoF BRCA1/2 mutations by cancer type modestly increased with their increasing mutational burden, while this was much more pronounced for BRCA1/2 variants of uncertain significance. e) Population frequency comparisons are shown between the study cohort and gnomAD for allele frequencies (AF) of BRCA1/2 germline pathogenic and likely pathogenic (P/LP) alleles and VUS (dark and light blue, respectively). Left, all alleles; center, only P/LP alleles; right, comparison between the study cohort and the germline results from the TCGA cohort. Ashkenzai Jewish (ASJ) founder BRCA1/2 alleles are shown (see legend). f) As in panel (e) for only the ASJ sub-populations. g) As in panels (e-f) but for the non-ASJ white subpopulation. NFE, non-Finnish European. h) The prevalence of homozygous deletions in BRCA1 or BRCA2 in affected cancer types. Count of affected tumors in parentheses, inset is the fraction of all homozygous deletions of either gene. i) Average age of first cancer diagnosis for BRCA1/2 germline carriers compared to those patients lacking any pathogenic germline alteration (germline WT) in BRCA-associated cancer types and all other cancer types. Error bars are 95% confidence interval (CI), asterisk reflects significance (two-sided Wilcoxon test, p-value<0.01; NS, not significant). j) Percent of BRCA1/2 germline carriers with multiple independent cancer diagnoses compared to germline WT patients. Error bars are 95% CIs, asterisk is p-value=0.02, chi-square test. k) The fraction of all germline pathogenic or somatic LoF alterations in BRCA1 versus BRCA2 (in non-hypermutated tumors).

Extended Data Fig. 2:. BRCA1/2 zygosity.

a) Diagrammatic representation of the integration of allele-specific copy number with purity and mutant allele frequencies to determine the zygosity of the germline pathogenic allele in the corresponding tumor (and the mechanism of its selection; CN-LOH, copy-neutral LOH). b) In only a subset of cases of low tumor cell content (<30%) does the LOH inference become increasingly analytically challenging (increasing rate of indeterminant calls). c) The percent of cases with LOH affecting the germline pathogenic or somatic LoF BRCA1/2 mutations (as labeled) as a function of tumor purity (see Methods). While somatic mutant allele frequencies are impacted by tumor purity, this does not affect the sensitivity for LOH detection for germline variants and only affects sensitivity for LOH of somatic mutations in tumors of <30% purity. d) In tumors with benign germline variants in BRCA1 and BRCA2, the ratio of zygosity changes affecting the WT or mutant BRCA1/2 allele is approximately 0.5, indicating neutral selection. By contrast, the rate of zygosity changes leading to loss of the WT allele in patients with germline pathogenic BRCA1 or BRCA2 mutations (>80%) is consistent with selective pressure for biallelic inactivation. e) Integrating all measurable sources of biallelic inactivation (inset, somatic sequence variants as the source of second hits to WT BRCA1/2), the percent of tumors by cancer type harboring a biallelic BRCA1 or BRCA2 loss. f) The rate of biallelic inactivation of BRCA1 versus BRCA2 in patients with germline pathogenic or somatic LoF mutations (in hypermutated and non-hypermutated tumors). P-values as indicated, two-sided Fisher’s exact test. NS, not significant. g) The rate of loss of WT BRCA1 or BRCA2 (LOH) in patients with germline deleterious BRCA1 or BRCA2 mutations compared to rare benign variants in either gene in BRCA-associated cancer types and in those not conventionally associated with BRCA germline carriers. Asterisks reflects significance (Fisher’s exact test). h) The rate of biallelic inactivation of BRCA1/2 in patients with germline pathogenic or somatic LoF mutations pan-cancer as a function of primary or metastatic specimen type. At right, the four BRCA-associated cancer types are shown individually. P-value as indicated, two-sided Fisher’s exact test. N.S. is not significant. Error bars are 95% CIs. i) The rate of LOH spanning germline or somatic mutant BRCA1 and BRCA2 in breast cancers (colored as in Fig. 2B–C) as well as other somatically mutated tumor-suppressor genes. Error bars are binomial CIs.

Extended Data Fig. 3:. Somatic loss of the pathogenic germline BRCA1/2 allele.

a) Schematic representation of the different allelic configurations that would lead to the retention or loss of a germline allele in the presence of a somatically mutated tumor suppressor gene (TSG) responsible for driving biallelic inactivation. b) Among tumors with loss of the pathogenic germline allele (in either BRCA1 or BRCA2, as indicated), the pattern of somatic mutations in known TSGs on their respective chromosomes (TP53 and NF1 are encoded on chromosome 17 on which BRCA1 also appears, while RB1 is encoded on chromosome 13 on which BRCA2 also appears) arising in the same tumors and in trans with, and presumed to drive the loss of, the germline allele. c) In a representative EML4-ALK positive lung adenocarcinoma diagnosed in a BRCA1 E23Vfs*17 carrier, LOH preceding whole-genome doubling spanned chromosome 17 encoding TP53 R248Q arising in trans with the mutant BRCA1 allele. Dark and light blue represent the major and minor copy number at the indicated loci. d) Somatic mutant allele factions (for case in panel C) are consistent with deletion of the allele harboring the BRCA1 founder mutation as compared to the observed and expected values for clonal heterozygous somatic mutations (RAD50) or biallelic inactivation of mutant TP53 (tumor purity is Φ). The selective pressure for biallelic TP53 inactivation driven by the initial R248Q mutation likely precipitated the subsequent heterozygous loss of the WT TP53 allele, leading to deletion of the BRCA1 pathogenic mutation and retention of the BRCA1 WT allele, indicating that mutant BRCA1 was likely dispensable for its pathogenesis.

Extended Data Fig. 4:. HRD phenotype in BRCA1/2-mutant cancers characterized by WES.

a) Total number of prospectively sequenced cases by cancer type for which exome re-sequencing was obtained. Abbreviations as in Extended Data Fig. 1; PNS, peripheral nervous system. b) The distribution of cancer types among BRCA1/2-mutant (germline or somatic) cases with exome re-sequencing data. c) The proportion of BRCA1/2-mutant cases with exome re-sequencing data by germline or somatic mutational origin. d) The somatic single-nucleotide mutational signature 3 of HRD, and e) the DNA copy number-based LST metric of HRD as inferred from exome sequencing data are shown as a function affected cancer types (left) and BRCA1/2 mutation origin and zygosity (right, see legend at bottom of panel E) as in main text Fig. 3. Asterisks reflect p-values<0.01, 10⁻¹⁰, and 10⁻²⁰ respectively, two-sided Student’s t-test. The individual metrics are highly correlated with the composite HRD score (rho=0.89, p-value=1e-270; see Methods) and consequently the qualitative results based on lineage, mutational origin and zygosity are similar. f) The rate of BRCA1 promoter methylation in ovarian, breast, and other cancer types (no evidence of BRCA2 silencing via promoter methylation was apparent). Inset, BRCA1 germline mutations and promoter methylation leading to BRCA1 silencing are mutually exclusive in affected cancers, indicating that heterozygous BRCA1-mutant tumors typically do not acquire biallelic inactivation via epigenetic silencing of the remaining allele. Epigenetic silencing is therefore unlikely to fully explain the modest HRD phenotype in heterozygous mutant tumors (Fig. 3B). Both germline and somatic mutational data and DNA methylation data was acquired from The Cancer Genome Atlas (see Methods). g) The composite measure of HRD in pan HR-wildtype tumors (light gray) and in tumors with either germline or somatic BRCA1 or BRCA2 mutations (dark gray) grouped by BRCA-associated cancer types (dark red: breast, ovary, pancreas, prostate) versus other cancer types (red), and tumors with somatic hypermutation (light red, see legend at bottom). Significant differences as indicated, two-sided Student’s t-test. NS, not significant. h) Same as in panel G and main text Fig. 3c, grouped by a combination of lineage, origin, and zygosity (see legend).

Extended Data Fig. 5:. Intra-individual BRCA phenotypic divergence.

Exome sequencing of two cancer diagnoses in a founder BRCA2 S1982Rfs*22 germline carrier revealed two independent and clonally unrelated cancer, one an HR-deficient serous ovarian cancer (left) with loss of WT BRCA2, the other a co-incident cholangiocarcinoma with intact WT BRCA2 (right). The latter had a different pattern of somatic abnormality and lacked any evidence of HRD (top) despite the shared germline pathogenic BRCA2 allele.

Extended Data Fig. 6:. Tumor mutational burden by BRCA1/2 genotype.

The somatic mutational burden of tumors as a function of cancer type, BRCA1/2 mutation origin, and somatic BRCA1/2 zygosity. Statistically significant differences among the indicated comparisons are shown. Two-sided Student’s t-test. NS, not significant. Error bars are the 95% confidence intervals.

Extended Data Fig. 7:. BRCA1/2 mutations attributable to other mutational signatures.

The somatic mutations in each of the evaluable cancers in main Fig. 3D in which a BRCA1 or BRCA2 somatic heterozygous mutation arose in a motif consistent with an alternative non-HRD mutational signature. The mutation (trinucleotide context, base change, and protein annotation) is indicated in each case as is its cancer type.

Extended Data Fig. 8:. PARP inhibitor therapy by BRCA1/2 mutation origin and zygosity.

A single PARP inhibitor outcome analysis of all four BRCA genotypes as independent classes (BRCA1/2 mutational origin and zygosity) with BRCA-associated cancer types (as in main text Fig. 4). All four classes of BRCA-mutant patients (see legend, as indicated) achieve significantly greater clinical benefit to PARP inhibitor therapy than do treated patients with WT BRCA tumors [BRCA1/2-mutant classes are germline carrier somatic heterozygous (HR=0.39, 0.21–0.72, p-value=0.003); germline carrier, somatic biallelic (HR=0.5, 0.35–0.72, p-value=2e-4); somatic heterozygous LoF (HR=0.5, 0.26–0.95, p-value=0.03); and somatic LoF biallelic (HR=0.34, 0.16–0.72, p-value=0.005).

Fig. 1:. The prevalence and origins of BRCA1/2 mutations.

a) Prevalence and type of germline pathogenic and somatic mutations in BRCA1 and BRCA2. Hypermutated tumors considered separately. b) The percent of BRCA1/2-mutant patients with a BRCA-associated cancer type (breast, ovary, prostate, pancreas) versus all other cancer types. c) BRCA alteration rates by gene and cancer type. Error bars are binomial confidence intervals (CIs), asterisks are p-value<0.05 for differences between BRCA1 and BRCA2 per tumor type, McNemar’s chi-square. Bottom, distribution of BRCA1/2 alteration types. d) Percent of patients by cancer type with germline or somatic BRCA1/2 alterations (red; p-value<0.05, enrichment for germline or somatic, McNemar’s chi-square).

Fig. 2:. Lineage variation in selection for BRCA1/2 biallelic inactivation.

a) The rate of LOH in BRCA1/2 pathogenic germline carriers [blue; diamond, biallelic inactivation via any mechanism (66%)], somatic LoF BRCA1/2 mutations, or TP53 oncogenic mutations (red). In gray, the background rate of LOH spanning benign germline variants or somatic passenger mutations (see Methods, asterisks are p-values < 0.01, 10⁻¹⁰, or 10⁻¹⁰⁰, respectively). NS, not significant. In all panels, error bars are binomial CIs. b) The rate of LOH in germline carriers compared to benign variants in BRCA-associated and select non-BRCA-associated cancer types. Asterisks reflects significance (two-sided Fisher’s exact test). c) Cancer type-specific rates of biallelic inactivation by mutation origin (bottom) and mechanism thereof (top). Dotted line, background rate of LOH pan-cancer. d) Percent of BRCA1/2 germline carriers that lose the pathogenic allele somatically in the indicated cancer types (p-value=0.003, two-sided Fisher’s exact test).

Fig. 3:. BRCA phenotypes are tumor lineage and zygosity-dependent.

a) The composite measure of HRD in pan-HR wildtype tumors versus those with BRCA1/2 germline or somatic mutations stratified by cancer type (BRCA-associated, non-BRCA-associated, and hypermutated tumors; see legend at right). Asterisks are p-values < 0.01, 10⁻¹⁰, and 10⁻²⁰ respectively, two-sided Student’s t-test. NS, not significant. b) As in panel a, grouped by mutation origin and zygosity (see legend). All individual comparisons with pan-HR wildtype tumors unless indicated with individual p-values. c) Same as in panels a-b, but grouped by a combination of lineage, origin, and zygosity (see legend). d) Tumors with somatic BRCA1/2 mutations with a dominant non-HRD mutational signature indicating an alternative mechanism of pathogenesis (0–5% HRD, which does not exceed the background rate in pan-HR wildtype tumors, Supplementary Table 6). Bottom, the likelihood the BRCA1/2 mutation was induced by the indicated signature (position and trinucleotide context indicative of the signature motif). Only samples with a dominant signature of known etiology are shown.

Fig. 4:. Context-specific therapeutic sensitivity of BRCA1/2-mutant tumors.

a) Left, clinical benefit to PARP inhibitor therapy in patients with BRCA-associated cancer types with and without BRCA1/2 mutations (germline or somatic) (HR 0.58, 95% CI 0.46–0.73, log-rank p-value=3.7e-6). Right, patients with all other cancer types (HR 1.02, 95% CI 0.6–1.7, log-rank p-value=0.98). b) In BRCA-associated cancer types, clinical benefit to PARP inhibition in patients with somatic LoF versus germline pathogenic BRCA1/2 alterations. c) As in panel (b) but comparing clinical benefit in heterozygous versus biallelic BRCA1/2-mutant BRCA-associated cancers. d-e) Event-free survival from the start of the first line of immune checkpoint blockade therapy in patients pan-cancer with or without BRCA1/2 germline or somatic mutations (HR=0.99, p-value=0.9 for non-hypermutated tumors). Multivariable model includes tumor mutational burden (>75^th percentile) and affected cancer type.