Longitudinal multi-omics study of palbociclib resistance in HR-positive/HER2-negative metastatic breast cancer

Abstract

Background: Cyclin-dependent kinase 4/6 inhibitor (CDK4/6) therapy plus endocrine therapy (ET) is an effective treatment for patients with hormone receptor-positive/human epidermal receptor 2-negative metastatic breast cancer (HR+/HER2- MBC); however, resistance is common and poorly understood. A comprehensive genomic and transcriptomic analysis of pretreatment and post-treatment tumors from patients receiving palbociclib plus ET was performed to delineate molecular mechanisms of drug resistance.

Methods: Tissue was collected from 89 patients with HR+/HER2- MBC, including those with recurrent and/or metastatic disease, receiving palbociclib plus an aromatase inhibitor or fulvestrant at Samsung Medical Center and Seoul National University Hospital from 2017 to 2020. Tumor biopsy and blood samples obtained at pretreatment, on-treatment (6 weeks and/or 12 weeks), and post-progression underwent RNA sequencing and whole-exome sequencing. Cox regression analysis was performed to identify the clinical and genomic variables associated with progression-free survival.

Results: Novel markers associated with poor prognosis, including genomic scar features caused by homologous repair deficiency (HRD), estrogen response signatures, and four prognostic clusters with distinct molecular features were identified. Tumors with TP53 mutations co-occurring with a unique HRD-high cluster responded poorly to palbociclib plus ET. Comparisons of paired pre- and post-treatment samples revealed that tumors became enriched in APOBEC mutation signatures, and many switched to aggressive molecular subtypes with estrogen-independent characteristics. We identified frequent genomic alterations upon disease progression in RB1, ESR1, PTEN, and KMT2C.

Conclusions: We identified novel molecular features associated with poor prognosis and molecular mechanisms that could be targeted to overcome resistance to CKD4/6 plus ET.

Trial registration: ClinicalTrials.gov, NCT03401359. The trial was posted on 18 January 2018 and registered prospectively.

Keywords: Advanced breast cancer; Drug resistance; Gene expression profiling; Genomic profile; Homologous recombination repair deficiengy (HRD); Palbociclib.

Fig. 1

Identification of prognostic markers. A Overview of the study design and data analysis. H&E, hematoxylin and eosin; HRD, homologous recombination deficiency. B Consort diagram showing patient enrollment and biopsy collection. QC, quality control. NGS profiling statuses for four patients are not shown: OT-only (n = 1), OT/PD (n = 1), and PD-only (n = 2). Forest plots of clinical variables (C) and molecular features (D) significantly associated with PFS. HR based on PFS calculated using univariate Cox regression analysis and variables with log-rank P-value < 0.05 considered significantly different in PFS. Continuous variables divided into high and low groups based on the median. C AI: letrozole/letrozole + GnRH/exemestane + GnRH. PR, progesterone receptor; M1, palliative treatment; ILC, invasive lobular carcinoma; IDC, invasive ductal carcinoma. D Signature: COSMIC Mutational Signature (version 2). TMB, tumor mutation burden; PAM50, intrinsic breast cancer subtype; non-luminal, HER2-enriched, basal, or normal-like subtype. E Correlogram (center) among clinical and molecular features significantly associated with PFS shows clusters of highly correlated covariates. Averages of variable importance for increased risk of progression (vertical right) based on 500 random resamples of survival elastic net models (75% training sets) show TP53 status, HRD (S3), and nuclear grade as the most important predictors of progression. In survival elastic net multivariate models, variable importance value is absolute standardized beta estimates

Fig. 2

HRD-high tumors co-occurring with TP53 mutations are associated with worse PFS. A Unsupervised clustering of genomic features. HRD cluster: classification of tumors harboring higher (HRD-H) or lower (HRD-L) level of HRD genomic scar features. S2 and S13 (APOBEC): mutation signatures 2 and 13 linked to APOBEC enzyme activity. S3 (HRD): mutation signature linked to homologous recombination deficiency. CYT score: cytolytic activity score calculated from mRNA expression levels of GZMA and PRF1. CIN, chromosome instability; TAI, telomeraic allelic imbalance; LOH, loss-of-heterozygosity; LST, large-scale transitions. Subtype: PAM50 subtype classification. B HRD-H cluster significantly enriched in BRCA1/2 mutations (Fisher’s exact test: p = 92.e−05). C HRD-H cluster significantly enriched in luminal B subtype (Fisher’s exact test: p = 0.0108436). D, E HRD-H cluster significantly enriched in HRD index (D) and mutation signature S3 (E) in the overall cohort and within luminal A and luminal B subtypes (Wilcoxon). F HRD-high cluster significantly associated with shorter PFS. G Kaplan-Meier plots comparing PFS between the four groups of baseline samples with different mutation statuses for TP53 and BRCA1/2. HR and 95% CI shown in parentheses. BRCA.mut+TP53.mut: co-occurring BRCA1/2 pathogenic mutation and TP53 somatic mutations. H Kaplan-Meier plots comparing PFS between the four groups of baseline samples with different statuses of HRD cluster and TP53 mutation. WT: TP53 wild type and HRD-Low. TP53.mut: TP53 mutation and HRD-Low. HRD-H: HRD-High and TP53 wild type. HRD-H+TP53.mut: TP53 somatic mutation and HRD-High

Fig. 3

Post-treatment enrichment of resistance markers. The non-luminal A subtype (A), HRD-H cluster (B), and proliferative cluster (C) were enriched in PD compared to baseline (Fisher’s exact test: p = 0.0084 for subtype; p = 0.054 for HRD cluster; p = 0.035 for proliferative cluster). Comparing the changes in HRD index, proliferative index, and S13 mutation signature at BL vs. PD among all samples (D) and longitudinally paired samples (E). The non-luminal A subtype (A), HRD-H cluster (B), and proliferative cluster (C) were enriched in PD compared to baseline (Fisher’s exact test: p = 0.0084 for subtype; p = 0.00314 for HRD cluster; p = 0.06351 for proliferative cluster). Comparing the changes in HRD index, proliferative index, and S13 mutation signature at BL vs. PD among all samples (D) and longitudinally paired samples (E). F Sankey diagram showing the switching of subtypes from luminal A at BL into HER2E or luminal B subtypes at PD. Comparing the changes in E2F targets signature (G), estrogen response early signature (H), and S13 mutation signature (I) between paired BL and PD tumors among the three groups of patients. To-HER2E: subtypes switched to HER2E at PD. To-LumB: subtypes switched to luminal B at PD. No-Switch: subtypes remained the same between BL and PD. GSVA: signature scored calculated by gene set variation analysis

Fig. 4

Landscape of PD-specific genomic alterations. A Stacked bar plots comparing the prevalence of genomic alterations at BL vs. PD for key BC genes. Colors represent the different genomic alterations including WT, missense, frameshift, inframe deletion, nonsense (splice site mutation, germline mutation) copy number amplification, copy number deletion, fusion, and mixed, indicating the tumor harbored multiple alterations. B Oncoprint of the mutational profile of selected genes for patients with paired baseline/on-treatment and PD samples. Paired samples for the same patient grouped together to highlight PD-specific alterations. Patients ordered by PFS as indicated by the track above the oncoprint. Colors represent the different mutation types. Stacked bar plots and number on the right show the number of patients with PD-specific alterations, with the percentage in parenthesis indicating the frequency of PD-specific alternations in 21 patients. C PD-specific RB1 mutations from our cohort in comparison with the spectrum of RB1 mutations reported in the PALOMA-3 study. We observed 6 mutations from 23.8% (5/21) of patients. Comparing longitudinal changes in RB1 TARGETS (D) and ESTROGEN RESPONSE EARLY signatures (E) between paired BL and PD tumors among the three groups. RB1 LOF: patients harboring PD-specific RB1 loss-of-function genomic alterations at PD. ESR1 GOF: patients harboring PD-specific ESR1 gain-of-function genomic alterations at PD. Other: all other patients with paired BL and PD samples. Patient BRO7F-093 harbored acquired mutations in both RB1 and ESR1 and was included in the “RB1 LOF” group in D and “ESR1 GOF” group in E. RB1 TARGETS: EGUCHI CELL CYCLE RB1 TARGETS gene set from mSigDB v5.2