Tracking evolution of aromatase inhibitor resistance with circulating tumour DNA analysis in metastatic breast cancer

Abstract

Background: Selection of resistance mutations may play a major role in the development of endocrine resistance. ESR1 mutations are rare in primary breast cancer but have high prevalence in patients treated with aromatase inhibitors (AI) for advanced breast cancer. We investigated the evolution of genetic resistance to the first-line AI therapy using sequential ctDNA sampling in patients with advanced breast cancer.

Patients and methods: Eighty-three patients on the first-line AI therapy for metastatic breast cancer were enrolled in a prospective study. Plasma samples were collected every 3 months to disease progression and ctDNA analysed by digital droplet PCR and enhanced tagged-amplicon sequencing (eTAm-Seq). Mutations identified in progression samples by sequencing were tracked back through samples before progression to study the evolution of mutations on therapy. The frequency of novel mutations was validated in an independent cohort of available baseline plasma samples in the Study of Faslodex versus Exemestane with or without Arimidex (SoFEA) trial, which enrolled patients with prior sensitivity to AI.

Results: Of the 39 patients who progressed on the first-line AI, 56.4% (22/39) had ESR1 mutations detectable at progression, which were polyclonal in 40.9% (9/22) patients. In serial tracking, ESR1 mutations were detectable median 6.7 months (95% confidence interval 3.7-NA) before clinical progression. Utilising eTAm-Seq ctDNA sequencing of progression plasma, ESR1 mutations were demonstrated to be sub-clonal in 72.2% (13/18) patients. Mutations in RAS genes were identified in 15.4% (6/39) of progressing patients (4 KRAS, 1 HRAS, 1 NRAS). In SoFEA, KRAS mutations were detected in 21.2% (24/113) patients although there was no evidence that KRAS mutation status was prognostic for progression free or overall survival.

Conclusions: Cancers progressing on the first-line AI show high levels of genetic heterogeneity, with frequent sub-clonal mutations. Sub-clonal KRAS mutations are found at high frequency. The genetic diversity of AI resistant cancers may limit subsequent targeted therapy approaches.

Keywords: ESR1; KRAS; breast cancer; ctDNA.

Figure 1

PlasmaDNA AI study of sequential plasma DNA sampling during first line aromatase inhibitor therapy for advanced breast cancer. (A) Consort diagram of plasma samples analysed for ESR1 mutations on the plasmaDNA AI study. (B) Baseline characteristics of patients in the PlasmaDNA AI study.

Figure 2

Evolution of ESR1 mutations during aromatase inhibitor (AI) therapy. (A–D) Mutation tracking in ctDNA collected during first line AI therapy. Data from four patients with ESR1 subclonal mutations detectable in ctDNA tracked until clinical progression. Allele fractions are shown as determined by sequencing. TP53 mutation in grey likely to have arisen from Clonal Haematopoesis of Indeterminate Potential (CHIP). (E) Percentage of cases with monoclonal (59.1%) or polyclonal ESR1 mutations (40.9%).

Figure 3

Error corrected ctDNA sequencing of plasma samples taken after progression on the first-line aromatase inhibitor (AI). Mutations identified in plasma DNA by eTAm-Seq error corrected sequencing, with ESR1 mutation analysis by ddPCR. Discordant cases for ESR1 between ddPCR ard ctDNA sequencing had lower mutant copies per ml in ddPCR compared with concordant cases (median 14.3 versus 51.5, respectively, P = 0.048 Mann–Whitney U test) and likely represent very low levels of mutant copies and random sampling. 8037 also had FGFR1 and ERBB2 amplification Identified. Of 36 progression plasma samples sequenced, 25 with mutations are displayed, 11 plasma samples with no mutations detected are not displayed. Numbers in boxes represent allele fraction for indicated gene. Where there are multiple mutations detected in the same gene, indicating polyclonality (P), aggregate allele fractions are given.

Figure 4

Lead time to development of ESR1 mutations. Serial tracking before progression, ESR1 mutations were detectable in plasma median 6.7 months [95% confidence interval (CI) 3.7–NA] before clinical progression.

Figure 5

Evolution of KRAS mutations during the first-line aromatase inhibitor (AI) therapy. (A–C) Mutation tracking in ctDNA collected during first line AI therapy. Data from three patients with KRAS subclonal mutations detectable in ctDNA tracked until clinical progression. Allele fractions are shown as determined by ddPCR. Patient B had an ALK mutation detected on sequencing at progression with an allele faction of 0.07. In two patients with KRAS mutations detected at progression primary tumour was available, with the KRAS mutation being undetectable in both patients.

Figure 6

Independent validation of KRAS mutations in baseline plasma from the SoFEA study. (A) Progression-free survival (PFS) in SoFEA by KRAS mutation status. HR, hazard ratio. (B) Overall survival (OS) in SoFEA by KRAS mutation status.