Hybrid capture-based genomic profiling of circulating tumor DNA from patients with estrogen receptor-positive metastatic breast cancer

Abstract

Background: Genomic changes that occur in breast cancer during the course of disease have been informed by sequencing of primary and metastatic tumor tissue. For patients with relapsed and metastatic disease, evolution of the breast cancer genome highlights the importance of using a recent sample for genomic profiling to guide clinical decision-making. Obtaining a metastatic tissue biopsy can be challenging, and analysis of circulating tumor DNA (ctDNA) from blood may provide a minimally invasive alternative.

Patients and methods: Hybrid capture-based genomic profiling was carried out on ctDNA from 254 female patients with estrogen receptor-positive breast cancer. Peripheral blood samples were submitted by clinicians in the course of routine clinical care between May 2016 and March 2017. Sequencing of 62 genes was carried out to a median unique coverage depth of 7503×. Genomic alterations (GAs) in ctDNA were evaluated and compared with matched tissue samples and genomic datasets of tissue from breast cancer.

Results: At least 1 GA was reported in 78% of samples. Frequently altered genes were TP53 (38%), ESR1 (31%) and PIK3CA (31%). Temporally matched ctDNA and tissue samples were available for 14 patients; 89% of mutations detected in tissue were also detected in ctDNA. Diverse ESR1 GAs including mutation, rearrangement and amplification, were observed. Multiple concurrent ESR1 GAs were observed in 40% of ESR1-altered cases, suggesting polyclonal origin; ESR1 compound mutations were also observed in two cases. ESR1-altered cases harbored co-occurring GAs in PIK3CA (35%), FGFR1 (16%), ERBB2 (8%), BRCA1/2 (5%), and AKT1 (4%).

Conclusions: GAs relevant to relapsed/metastatic breast cancer management were identified, including diverse ESR1 GAs. Genomic profiling of ctDNA demonstrated sensitive detection of mutations found in tissue. Detection of amplifications was associated with ctDNA fraction. Genomic profiling of ctDNA may provide a complementary and possibly alternative approach to tissue-based genomic testing for patients with estrogen receptor-positive metastatic breast cancer.

Keywords: ER; ESR1; ctDNA; genomic profiling; liquid biopsy; metastatic breast cancer.

Figure 1.

Genomic alterations in ctDNA from patients with ER+ breast cancer and comparisons with tissue. (A) GAs identified in 197 cases of ER+/HER2− BC. Percent of cases altered is indicated. (B) GAs identified in 28 cases of ER+/HER2+ BC. (C) Comparison of the most frequently mutated (top panel) or amplified (bottom panel) genes observed in ctDNA in this study (N = 254) with tissue-based genomic profiling of ER+ BC. Datasets from ER+ BC tissue used for comparison were from the FM database (N = 851) and published studies of tissue from early BC (eBC, TCGA [1]: N = 594) and mBC (Lefebvre et al. [3]: N = 143; Fumagalli et al. [2]: N = 182). Data from [1, 3] were extracted from cBioPortal. Black dots represent genes that were not assessed in [2]. (D) Concordance between GAs found in ctDNA and matched tumor tissue from 14 patients. Days between ctDNA and tissue collection are shown. The ctDNA fraction was estimated using MSAF. Concordant/shared GAs are in blue, GAs found in tissue only are in grey, and GAs found in ctDNA only are in red. For samples with multiple unique mutations in a gene (patient-5 and patient-8), the number of mutations is shown.

Figure 2.

Landscape of ESR1 alterations in ctDNA. (A) Graph represents the number of cases with each ESR1 GA. The percent indicate the frequency of each ESR1 GA relative to all 131 ESR1 GA identified. AMP, amplification; RE, rearrangement. (B) Percent of cases with 1, 2, 3, or 4 ESR1 GAs. (C) Compound mutations in ESR1: in case-1 all instances of L536F and D538G were observed on the same read in cis. In case-2 ESR1 c.1609T>A (Y537N) was observed alone in the majority of reads, and ESR1 c.1609T>A and c.1610A>G were seen together as compound mutations in a portion of reads to generate ESR1 Y537S. (D) ESR1 variants of unknown significance identified in this study. (E) ESR1 rearrangements identified in this study. Numbered boxes represent exons and numbers above indicate amino acid position. Rearrangement break points in ESR1 were in exon 4 or exon 5. (F) Assessment of GAs that co-occur with ESR1 GA: frequency of ESR1-altered cases (N = 80) with alterations in the genes indicated. ‘Multiple’ represents cases harboring multiple classes of genomic alteration.