Real-World Use of Highly Sensitive Liquid Biopsy Monitoring in Metastatic Breast Cancer Patients Treated with Endocrine Agents after Exposure to Aromatase Inhibitors

Abstract

Endocrine-resistant, hormone receptor-positive, and HER2-negative (HR+/HER2-) metastatic breast cancer (mBC) is largely governed by acquired mutations in the estrogen receptor, which promote ligand-independent activation, and by truncal alterations in the PI3K signaling pathway, with a broader range of gene alterations occurring with less prevalence. Circulating tumor DNA (ctDNA)-based technologies are progressively permeating the clinical setting. However, their utility for serial monitoring has been hindered by their significant costs, inter-technique variability, and real-world patient heterogeneity. We interrogated a longitudinal collection of 180 plasma samples from 75 HR+/HER2- mBC patients who progressed or relapsed after exposure to aromatase inhibitors and were subsequently treated with endocrine therapy (ET) by means of highly sensitive and affordable digital PCR and SafeSEQ sequencing. Baseline PIK3CA and TP53 mutations were prognostic of a shorter progression-free survival in our population. Mutant PIK3CA was prognostic in the subset of patients receiving fulvestrant monotherapy after progression to a CDK4/6 inhibitor (CDK4/6i)-containing regimen, and its suppression was predictive in a case of long-term benefit with alpelisib. Mutant ESR1 was prognostic in patients who did not receive concurrent CDK4/6i, an impact influenced by the variant allele frequency, and its early suppression was strongly predictive of efficacy and associated with long-term benefit in the whole cohort. Mutations in ESR1, TP53, and KRAS emerged as putative drivers of acquired resistance. These findings collectively contribute to the characterization of longitudinal ctDNA in real-world cases of HR+/HER2- mBC previously exposed to aromatase inhibitors and support ongoing studies either targeting actionable alterations or leveraging the ultra-sensitive tracking of ctDNA.

Keywords: breast cancer; ctDNA; endocrine resistance; liquid biopsy; precision medicine; real-world evidence.

Figure 1

Mutational landscape in baseline ctDNA. (A) Oncoprint plot show somatic alterations and clinical–pathological characteristics per patient. (B) Swimmer plot displaying individual progression-free survival and mutations in plasma at baseline and at progression. (C) Lolliplots showing the spectrum of detected mutations in PIK3CA, ESR1, and TP53. (D) Mutation prevalence across selected published studies including landmark trials and large patient cohorts. Abbreviations: PR, partial response; SD, stable disease; PD, progressive disease; NE, non-evaluable; #, number of mutations; arrows, patients with ongoing treatment; [19,20,21,22,23].

Figure 2

Concordance between dPCR and SafeSEQ NGS. (A) Inter-technique agreement in the detection of PIK3CA (green) and ESR1 (pink) mutations. (B) Venn diagrams displaying concordance for all analyzed samples (upper left, PIK3CA; down left, ESR1) and mutations with VAF < 1% (right). (C,D) Correlation of VAF and mutant molecules per mL in mutant samples. Green and red dots denote mutations in PIK3CA and ESR1, respectively. ***, p < 0.001.

Figure 3

cfDNA levels at baseline and early dynamics. (A) Increase in cfDNA levels at progression. (B,C) Prognostic and predictive value of baseline cfDNA levels. *, p < 0.05.

Figure 4

ctDNA levels at baseline. (A–C) Prognostic value of mutant PIK3CA in the whole cohort (A), patients who did not receive CDK4/6i while on study (B) and patients who had progressed to prior CDK4/6i and received fulvestrant monotherapy while on study (C). (D,E) Prognostic value of mutant ESR1 in the whole cohort (D) and in patients who did not receive CDK4/6i while on study (E). (F) Differential impact of ESR1 VAF on PFS. (G) Prognostic value of TP53 mutations.

Figure 5

ctDNA early dynamics. (A) Lack of a predictive role by early PIK3CA mutant VAF suppression (VAF T1 = 0). (B) ESR1 suppression is associated with an improved PFS. (C,D) ESR1 mutation dynamics from baseline (t0) to 8 ± 2 weeks into treatment (t1) to distinguish patients with upfront resistance (PFS < 4.5 months) or long-term benefit (PFS > 9 months). (E) Operational characteristics of ESR1 mutant VAF suppression at 9 and 4.5 months of treatment. NS, not significant; *, p < 0.05.

Figure 6

Clinical cases and subgroups of interest. (A–H) Clonal evolution under therapeutic pressure along with clinical vignettes showing the therapy received during the study period, the previous use of CDK4/6i, and the PFS attained. (I) The prevalence of PIK3CA, ESR1 (red), and TP53 (gray) alterations stratified by HER2-low or HER2-zero status in our cohort. (J,K) Grid displaying the therapy received during the study period and the mutation status of PIK3CA, ESR1, TP53, and KRAS at baseline and at progression in patients selected by their unexpectedly prolonged (J) or short (K) PFS. Shadowing denotes mutant status, white denotes non-mutant status, and gold denotes untested. Green: PIK3CA, Red: ESR1; Gray: TP53; Blue: ERBB2; Orange: KRAS.