Analysis of ESR1 and PIK3CA mutations in plasma cell-free DNA from ER-positive breast cancer patients

Abstract

Background: The measurement of ESR1 and PIK3CA mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive method to quickly assess and monitor endocrine therapy (ET) resistant metastatic breast cancer (MBC) patients.

Methods: The subjects of this retrospective study were a total of 185 plasma samples from 86 estrogen receptor-positive BC patients, of which 151 plasma samples were from 69 MBC patients and 34 plasma samples were from 17 primary BC (PBC) patients. We developed multiplex droplet digital PCR assays to verify the clinical significance of ESR1 and PIK3CA mutations both in a snapshot and serially in these patients.

Results: cfDNA ESR1 and PIK3CA mutations were found in 28.9% and 24.6 % of MBC patients, respectively. The relation between ESR1 or PIK3CA mutations and clinical features showed that ESR1 mutations occurred mostly in patients previously treated by ET, which was not the case for PIK3CA mutations. The analysis of the clinical impact of those mutations on subsequent lines of treatment for the 69 MBC patients revealed that both ESR1 and PIK3CA mutations detection were related to a shorter duration of ET effectiveness in univariate analysis but only for ESR1 mutations in multivariate analysis. The monitoring of cfDNA in a subset of 52 patients showed that loss of ESR1 mutations was related to a longer duration of response, which was not the case for PIK3CA mutations.

Conclusions: We have demonstrated the clinical significance of on-treatment ESR1 mutations both in a snapshot and serially in comparison with PIK3CA mutations.

Keywords: ESR1 mutations; PIK3CA mutations; acquired endocrine therapy resistance; cell-free DNA; estrogen receptor-positive metastatic breast cancer.

Figure 1

A. Representative ddPCR analysis of polyclonal ESR1 mutations. The presence of all three hotspot LBD ESR1 mutations (Y537S, Y537N, and D538G) was confirmed by uniplex assays (Supplementary Figure S1). In each plot, green dots represent HEX-labeled wild type DNA, blue dots represent FAM-labeled mutant DNA, and black dots are droplets with no DNA incorporated. B. Comparison of each LBD ESR1 mutation between uniplex and multiplex ddPCR assays from a validation subset of 26 women (62 blood samples). Abbreviations; ddPCR, droplet digital polymerase chain reaction; LBD, ligand binding domain; cfDNA.

Figure 2

A. Chart showing the percentage of ESR1 mutations and PIK3CA mutations in plasma cfDNA in this cohort. B. Comparison of the number of copies/μL between total ESR1 mutations and total PIK3CA mutations in plasma from a subset of 86 women (185 blood samples). Abbreviations; cfDNA, cell-free DNA; H1047x, H1047L/R/Y; E545x Q546x, E545V/G/A/Q/K Q546L/R/P/E/K; E542x, E542K/V; G1049x, G1049R/S.

Figure 3. Kaplan-Meier plots of the association of ESR1 mutations and PIK3CA mutations in cfDNA with duration of ET effectiveness in this cohort

These were verified by the log-rank test. A, B. Patients with detectable plasma ESR1 mutations (P < 0.0001) (A) and PIK3CA mutations (P = 0.0034) (B) showed significantly shorter duration of ET effectiveness in 69 MBC patients. C., D. Tracking analysis of cfDNA ESR1 mutations and PIK3CA mutations in 52 breast cancer patients with longitudinal samples. Patients were grouped into those who did not have any ESR1 or PIK3CA mutations over the course of treatment, those in whom ESR1 or PIK3CA mutations were maintained or acquired, and those in whom ESR1 or PIK3CA mutations disappeared in cfDNA. C. Patients in the loss of cfDNA ESR1 mutations group had a longer duration of ET effectiveness than patients with acquired or maintained numbers of cfDNA ESR1 mutations, but had a shorter duration of ET effectiveness than patients without mutations over the course of treatment (P < 0.0001). D. There was no statistically significant differences in these three groups; no PIK3CA mutations during treatment group, the loss of cfDNA PIK3CA mutations group, and the acquired or maintained numbers of cfDNA PIK3CA mutations group. Abbreviations: ET, endocrine therapy; cfDNA, cell-free DNA; MBC, metastatic breast cancer.

Figure 4

A.-D. Intrapatient changes of the numbers of copies/μL of ESR1 and PIK3CA mutations under treatment with each representative therapeutic drug in 21 patients with longitudinal data. A. Patients treated with AIs, B. Patients treated with SERDs, C. Patients treated with SERMs, D. Patients treated with EE2. Abbreviations; AI, aromatase inhibitor; SERD, selective estrogen receptor downregulator; SERM, selective estrogen receptor modulator; EE2, ethinyl estradiol.