Homologous recombination deficiency and hemizygosity drive resistance in breast cancer

Resistance to anticancer therapy often occurs through a diverse array of genomic mechanisms^1,2,3,4,5. The inherent unpredictability of these events challenges the ability to develop proactive strategies to pre-empt the emergence of resistance and improve patient outcomes. The role of germline pathogenic variants in predisposition to malignancies and shaping the initial somatic landscape of tumours is well established^6,7,8,9. This interplay is well exemplified in germline pathogenic variants in certain DNA damage repair genes^{10,11,12,13,14} that give rise to characteristic somatic allelic configurations and genomic instability in the form of homologous recombination deficiency (HRD)^15,16,17, a biology that has been successfully exploited with PARP inhibitors (PARPi)^18,19,20. However, despite our understanding of these initial events, the influence of germline pathogenic variants on the subsequent evolutionary life of a tumour remains poorly defined.

Clinically, the expanding landscape of approved targeted and lineage-directed therapies has introduced considerable uncertainty regarding the optimal treatment paradigm for patients with breast cancer with certain germline and somatic backgrounds. Given its dominant effect on initial cancer evolution, we posit that the germline background may equally influence tumour behaviour under therapeutic pressure, effectively directing the evolutionary trajectory of disease progression and resistance.

More broadly, defining how pre-treatment germline and somatic genomic context shapes the path of acquired therapy resistance could enable early interception strategies to prevent or delay the emergence of drug-resistant tumour clones. To elucidate the therapeutic relevance of germline–somatic interactions in a clinically meaningful setting, we performed an integrated analysis of germline and somatic genomic data paired with detailed clinical annotation, including treatment response, in a large cohort of patients with breast cancer.

To identify the interactions between germline pathogenic variants and somatic oncogenic alterations in breast cancer, we integrated detailed clinical annotation with prospectively collected sequencing data^21,22,23 based on 6,927 tumours from 5,881 patients with breast cancer (Memorial Sloan Kettering Cancer Center (MSK) cohort; Extended Data Fig. 1a and Supplementary Table 1). DNA derived from tumour tissue and blood as a source of germline DNA were each sequenced using an FDA-authorized clinical sequencing assay encompassing up to 506 cancer-associated genes, including germline analysis of 84 cancer predisposition genes^21,22,23. Genes of interest for the germline analysis included canonical members of the homologous recombination pathway^{10,11,12,13,14}: BRCA2 (2.9%, n = 161 patients), BRCA1 (2.6%, n = 142 patients), CHEK2 (1.6%, n = 87 patients), ATM (1.1%, n = 60 patients) and PALB2 (0.6%, n = 35 patients).

The clinicopathological characteristics of the germline-altered cancers and germline wild-type (gWT) cancers strongly reflected previously established patterns, suggesting that our cohort was representative of the broader population of patients with breast cancer (Table 1). Specifically, we observed a younger age of diagnosis in gBRCA1/2 carriers than in gWT. gBRCA1-associated breast cancers tended to be triple-negative²⁴ and high-grade invasive ductal carcinomas. Meanwhile, gBRCA2, gCHEK2 and gATM carriers typically had hormone receptor-positive and HER2-negative (HR⁺/HER2^–) disease (75.2%, 74.7% and 71.7%, respectively; Supplementary Table 2), consistent with previous studies^25,26.

Biallelic loss is often a necessary condition to observe a phenotypic impact in carriers of germline pathogenic variants in HRD-related genes, yet its incidence varies by gene, with higher rates of biallelic inactivation observed in high-penetrance genes^27,28. Our results confirmed that biallelic inactivation rates varied significantly across genes, ranging from 50.6% (n = 44) in gCHEK2 carriers to 77.5% (n = 110) and 75.8% (n = 122) for gBRCA1 and gBRCA2 carriers, respectively (Extended Data Fig. 1b). We also found lower frequency of loss of heterozygosity (LOH) in gPALB2 carriers (51.4%, n = 18), but a relatively higher frequency of ‘second-hit’ somatic mutations resulting in biallelic loss (33.3%, n = 6), concurring with previous literature²⁹. Confirming the associations between histological, demographic and genomic patterns with germline pathogenic variants establishes the clinical relevance of this clinicogenomic cohort.

We first sought to define patterns of mutual exclusivity or enrichment of somatic variants with germline pathogenic variants (germline–somatic interactions), in the context of breast cancer receptor subtype and zygosity (Fig. 1a). This analysis robustly validated previously reported enrichment of TP53 alterations in gBRCA1 carriers and mutual exclusivity of gATM and TP53 alterations^30,31 (Fig. 1b,c). Both findings were more pronounced in tumours exhibiting biallelic inactivation of the respective genes (Extended Data Fig. 2a,e). We further explored these observations by conducting germline–somatic interaction analyses stratified by breast cancer receptor subtypes. The gBRCA1–TP53 interaction was enriched in HR⁺/HER2^– tumours but not in triple-negative tumours, where TP53 variants are already highly prevalent in BRCA1 WT tumours, supporting TP53 loss of function (LoF) as a ubiquitous event in the oncogenesis of gBRCA1-driven breast cancers^32,33.

a, Study schema. Enrichment analysis was performed to identify somatic alterations more prevalent in patients with pathogenic germline variants of homologous recombination pathway genes (compared with ‘sporadic’ or gWT cases). FC, fold change; LCN, lesser copy number; TCN, total copy number. Schematic created in BioRender; Razavi, P. https://biorender.com/hv0bvbr (2025). b, Enrichment analysis of gATM (n = 60) compared with gWT (n = 5,094), as determined by Firth-penalized logistic regression with sample type and receptor status as covariates. Significant genes (blue) are defined by q < 0.10. c, As in panel b, but for comparison of gBRCA1 (n = 142) with gWT (n = 5,904).gBRCA1 tumours were enriched for somatic TP53 variants; OR = 2.80 (95% CI 1.70–4.59, q = 0.0009) and mutually exclusive with PIK3CA variants. d, As in panels b,c, but for comparison of gBRCA2 (n = 161) compared with gWT (n = 5,094). A subset analysis of HR⁺/HER2^– tumours (n_Total = 3,547, n_gBRCA2 = 121 and n_gWT = 3,426) was then performed. Somatic RB1 variants represented the most significant enrichment in gBRCA2 carriers among this subgroup; OR = 5.66 (95% CI 3.34–9.60, q = 4.7 × 10⁻⁹). e, Oncoprint showing mutations, copy number deletions and fusions in the indicated genes in gBRCA2 versus gWT. Receptor status, sample type and zygosity are annotated above.

In gBRCA2-driven breast cancers, RB1 somatic variants were significantly enriched, in stark contrast to their absence in gBRCA1-associated tumours (Fig. 1c–e and Extended Data Fig. 2b). This observation is notable, as gBRCA1 tumours are largely triple negative, a subtype typically enriched for RB1 alterations. Focusing on patients with HR⁺/HER2⁻ tumours (75.2% of gBRCA2) revealed an even higher enrichment of somatic RB1 alterations in gBRCA2 carriers. This analysis also uncovered an enrichment of MYC and AURKA amplifications. Of clinical relevance, RB1, MYC and AURKA alterations have all been implicated in resistance to CDK4/6 inhibition^34,35. Conversely, PIK3CA alterations were more enriched in gWT cancers than in gBRCA2 and gBRCA1-driven cancers. The receptor status and LOH-specific germline–somatic interactions are further displayed in Extended Data Fig. 2, and corresponding Oncoprints are detailed in Extended Data Fig. 3 (Supplementary Tables 3–7).

CDK4/6i combined with endocrine therapy (CDK4/6i + ET) represents the cornerstone of treatment for patients with metastatic and high-risk early-stage HR⁺/breast cancer^36,37,38, with RB1 loss established as a key mechanism of resistance to CDK4/6i³⁹. On the basis of our results demonstrating a significant enrichment of RB1 alterations in gBRCA2 carriers, we analysed the effect of gBRCA2 status on progression-free survival (PFS) in patients with HR⁺/HER2^– metastatic breast cancer (MBC) treated with CDK4/6i + ET. gBRCA2 pathogenic variants were associated with a significantly shorter PFS on CDK4/6i + ET in univariate and multivariate analyses (median PFS of 9.0 versus 15.6 months, multivariate hazard ratio (HR) = 2.17, 95% CI 1.60–2.96, P < 0.00001; Fig. 2a). Similar results were seen when the analysis was extended to all treatment lines, with consideration of ET partner and treatment line as covariates (HR = 1.97, 95% CI 1.55–2.51, P < 0.00001; Fig. 2b and Supplementary Table 8). LoF mutations in RB1 were rare in pre-treatment samples (2%), and exclusion of these cases did not alter the results.

a, PFS for patients treated with first-line CDK4/6i + ET by gBRCA2 status. Patients with gBRCA2 (n = 51) were compared with gWT (n = 926); P = 8.0 × 10⁻⁷. b, As in panel a, but depicting all lines of treatment. Patients with gBRCA2 (n = 80) were compared with gWT (n = 1,670); P = 2.9 × 10⁻⁸. c, PFS on first-line CDK4/6i + ET combinations by gBRCA2 status from the multi-institutional external validation cohort, which included 61 patients with the gBRCA2 pathogenic variant and 2,097 with gWT. HRs in panels a–c were estimated using Cox proportional hazard models (P values from two-sided Wald tests). No multiple comparisons adjustment was made. d, Swimmer’s plot of patients (n = 41) receiving PARPi (blue bar) after CDK4/6i + ET (red bar); HR_PARPi versus HR_CDK4/6i = 0.38 (95% CI 0.19–0.76, P = 0.0062), based on Cox proportional hazard model stratified by patient ID. Best response (complete or partial response) was compared for PARPi versus CDK4/6i + ET using a two-sided Fisher’s exact test; OR = 8.87 (95% CI 2.84–27.98, P = 0.00002). MD, physician; NE, non-evaluable. e,f, Two representative cases demonstrating short PFS on first-line (1L) CDK4/6i + ET and subsequent metabolic complete response (CR) and durable disease control on PARPi. 2L, second line; 3L, third line; AI, aromatase inhibitor; POD, progression of disease; PR, partial response. Black and yellow arrows denote radiographic tumour activity. g, Paired pre-CDK4/6i or post-CDK4/6i oncoprint by gBRCA2 status. The black rectangle denotes acquired variant. Two-sided Fisher’s exact test of RB1 LoF variant acquisition by gBRCA2 status; OR = 5.17 (95% CI 2.07–13.0, P = 0.0010).

We next validated our findings using an independent, nationwide clinicogenomic dataset containing manually curated patient-level outcomes data from both community oncology settings and academic medical centres^40,41. This analysis confirmed a strong association between gBRCA2 and shorter PFS on CDK4/6i + ET (median PFS of 12.0 versus 18.3 months, HR = 1.83, 95% CI 1.36–2.48, P < 0.00001; Fig. 2c) among the 2,158 patients with HR⁺/HER2^– MBC treated with first-line CDK4/6i + ET combinations. To evaluate whether gBRCA2 was associated with resistance specifically to CDK4/6i + ET, we expanded our analysis to assess the effect of gBRCA2 status on PFS of other common therapeutic modalities in breast cancer (Extended Data Fig. 4a). gBRCA2 status did not affect outcome on the vast majority of these therapies, nor did it significantly effect overall survival for patients starting on first-line CDK4/6i + ET. This demonstrates that gBRCA2 status is not a universal determinant of outcome but instead confers context-dependent relevance to specific therapies.

We further investigated the MSK cohort focusing on patients with HR⁺/HER2^– MBC who received a PARPi after progression on CDK4/6i (n = 41). Of note, despite being administered in later lines (median line of therapy = 3), HRD-directed therapy generally resulted in superior outcomes (Fig. 2d). PARPi treatment resulted in a significantly improved PFS compared with the preceding CDK4/6i regimen (HR = 0.38, 95% CI 0.19–0.76, P = 0.0062), with 73.2% of patients achieving a longer PFS on PARPi than on frontline CDK4/6i. This clinical benefit was more pronounced among the patients who had failed to respond to previous CDK4/6i + ET (Fisher’s exact test; OR = 8.87, 95% CI 2.84–27.98, P = 0.00002; Extended Data Fig. 4b). Among patients with evaluable imaging who did not discontinue therapy due to early toxicity (n = 38), PARPi achieved a partial or complete response in 84.2% (n = 32) of patients, compared with only 39.5% (n = 15) for previous CDK4/6i + ET (Extended Data Fig. 4c). Representative cases of rapid progression through first-line CDK4/6i + ET, followed by prolonged complete response to PARPi are highlighted in Fig. 2e,f. Collectively, these results provide a clinical rationale for prioritization of PARPi for this genomically defined subgroup of patients with breast cancer with expected poor outcomes on CDK4/6i-based combinations.

To investigate the implications of germline status on the development of RB1 loss, an established mechanism of CDK4/6i resistance, we assembled a large cohort of patients with paired tumour samples collected pre-CDK4/6i and post-CDK4/6i (1,312 tumour and 513 plasma circulating tumour DNA (ctDNA) samples from 546 patients). Genomic analysis of this paired pre-treatment and post-progression cohort demonstrated that acquired somatic RB1 LoF alterations were significantly more prevalent in gBRCA2 tumours versus gBRCA2 WT tumours (28.6% versus 7.1%, OR = 5.17, 95% CI 2.07–13.0, P = 0.0010; Fig. 2g). The clinical responses and evolutionary trajectories of patients treated with CDK4/6i are highlighted by two representative patients with gBRCA2 HR⁺/HER2^– MBC (Extended Data Fig. 4d,e). In both cases, acquired polyclonal RB1 LoF variants emerged post-CDK4/6i, underscoring a strong and specific evolutionary pressure for RB1 LoF mutations as a dominant mechanism of CDK4/6i resistance in these gBRCA2 tumours.

Consideration of the genomic architecture of BRCA2-driven tumours provides further insight into the acquisition of RB1 LoF alterations. BRCA2 and RB1 are syntenic on chromosome 13q, and previous work suggests that biallelic inactivation of BRCA2 in gBRCA2-driven tumours often occurs through loss of a large chromosomal segment inclusive of WT BRCA2 and RB1 alleles^{42,43,44,45,46,47}. Consistent with this, the pattern of RB1 LOH was influenced by gBRCA2 status in our cohort, demonstrating a co-occurrence of BRCA2 and RB1 LOH in gBRCA2 tumours compared with gWT (Fig. 3a). We validated this finding using an external cohort of 46 gBRCA2-associated primary breast cancers profiled with whole-exome sequencing (24 patients from the University of Pennsylvania Abramson Cancer Center and 22 from the Mayo Clinic). Consistently, 82.6% (n = 38) of tumours in this cohort demonstrated concurrent LOH of BRCA2 and RB1 (P < 0.0001; Extended Data Fig. 5). Although RB1 LOH was enriched in gBRCA2 tumours, it notably was also present in 34.9% of gWT tumours (n = 601 of 1,723 of evaluable cases), suggesting a broader relevance for this allelic state.