Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer

Abstract

Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AIs). We developed ultra high-sensitivity multiplex digital polymerase chain reaction assays for ESR1 mutations in circulating tumor DNA (ctDNA) and investigated the clinical relevance and origin of ESR1 mutations in 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies and was accurately assessed in samples shipped at room temperature in preservative tubes. ESR1 mutations were found exclusively in estrogen receptor-positive breast cancer patients previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy [hazard ratio, 3.1; 95% confidence interval (CI), 1.9 to 23.1; P = 0.0041]. ESR1 mutation prevalence differed markedly between patients who were first exposed to AI during the adjuvant and metastatic settings [5.8% (3 of 52) versus 36.4% (16 of 44), respectively; P = 0.0002]. In an independent cohort, ESR1 mutations were identified in 0% (0 of 32; 95% CI, 0 to 10.9) tumor biopsies taken after progression on adjuvant AI. In a patient with serial sampling, ESR1 mutation was selected during metastatic AI therapy to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI but are commonly selected by therapy for metastatic disease, providing evidence that mechanisms of resistance to targeted therapy may be substantially different between the treatment of micrometastatic and overt metastatic cancer.

Copyright © 2015, American Association for the Advancement of Science.

Figures

Figure 1. Assessment of ESR1 in circulating tumor DNA with multiplex digital PCR assays has analytical and clinical validity

A. High sensitivity multiplex digital PCR for ligand binding domain (LBD) ESR1 mutations. Representative digital PCR analysis of plasma DNA from two separate patients. Top panel: detection of an apparently monoclonal Y537N (c.1609T>A) mutation. Bottom panel: detection of polyclonal ESR1 mutations. The presence of all five mutations was confirmed by uniplex assays, with Y537C (c.1610A>G) and L536R (c.1607T>G) detectable in low amounts. In each plot, green dots represent VIC-labeled wild type DNA (except for the population labelled as “ghost,” which represents droplets with mutations different from those analyzed in the assay), blue dots represent FAM labeled mutant DNA, brown dots represent droplets containing both wild type and mutant DNA, and black dots are droplets with no DNA incorporated. B. Comparison of ESR1 mutation calling between contemporaneous tumor biopsies and ctDNA plasma samples from 31 patients with advanced breast cancer, with overall 97% agreement between tumor DNA and ctDNA analysis, Kappa 0.84 (95% CI 0.53-1.0). Detection of ESR1 in ctDNA has 100% positive predictive value for tumor ESR1 mutation status and 96.4% negative predictive value. C. Comparison of ESR1 mutation calling between repeat samples from the same 43 patients. 2 different tubes (1 EDTA tube and 1 cell free DNA Streck tube) were used at the same blood draw and processed entirely separately. There was 100% agreement between assays (Kappa 1.0, 95% CI not estimable), with exact concordance of the mutation called between samples. D. Correlation between mutation abundance (mutant copies per ml of plasma) assessed in EDTA and Streck tubes in ESR1 mutant plasma samples. r2=0.92; Pearson's correlation coefficient.

Figure 2. Detection of ESR1 mutations in plasma predicts lack of sensitivity to subsequent AI therapy

A. ESR1 ctDNA analysis by multiplex digital PCR in 171 patients with advanced breast cancer. ESR1 mutations are detected exclusively in plasma of patients with ER positive advanced breast cancer, p=0.0093 Chi Square test. B. Left. Profile of ESR1 mutations detected in ctDNA. Right Percentage of cases with apparently monoclonal (79%) or polyclonal (21%) ESR1 mutations. C. Progression-free survival on AI therapy after ctDNA analysis, for patients with ESR1 mutant and wild type ctDNA (HR 3.1, 95%CI 1.9-23.1, p=0.0041 log rank test).

Figure 3. ESR1 mutations are rarely acquired during adjuvant AI, and frequently during metastatic AI therapy

A. ESR1 ctDNA mutation rate split by timing of patients’ exposure to AI therapy, or no prior exposure to AI therapy. P<0.0001 Chi Square test overall, and p=0.0002 Chi Square test comparing metastatic AI only to prior exposure first in the adjuvant setting. B. PIK3CA ctDNA mutation rate, assessed with multiplex digital PCR assay, split by timing of patients’ exposure to AI therapy, or no prior exposure to AI therapy. Samples from 19 patients included in part A were not analyzed because plasma was exhausted. p=0.66, Chi squared test.

Figure 4. Validation and independent series confirm the importance of timing of prior AI exposure for ESR1 mutation selection

A. ESR1 mutation rate assessed only in patients with detection of a mutation other than ESR1 in plasma DNA. p=0.061 Chi Square test overall and p=0.035 adjuvant AI only vs metastatic AI only. B. Assessment of ESR1 mutation rate in an independent series of 49 breast tumor biopsies that had recurred after prior AI therapy. No ESR1 mutations were identified in breast tumor biopsies relapsing after adjuvant AI (0%, 95% CI 0-10.9%). C. Reassessment of a second independent series of ESR1 mutant positive cancers, with timing of prior AI therapy (9).

Figure 5. ESR1 mutations are selected to become the dominant clone in the cancer

A. Serial ctDNA analysis in a patient with plasma samples taken before and after exposure to AI for metastatic breast cancer. Tumor PIK3CA mutation is detected in both plasma samples, whilst ESR1 mutation is only detected after developing resistance to AI. OvS: ovarian suppression (goserelin, followed by bilateral salpyngo-oophorectomy); tam: tamoxifen. B. Relative abundance of ESR1 mutations in ctDNA compared to abundance of other tumor derived mutations detected in ctDNA. Below dotted line (0.25) suggests likely sub-clonal mutations.