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.


Introduction
Genomic changes that characterize primary breast cancer (BC) have been elucidated by extensive genomic profiling studies [1]. Comparative analyses of estrogen receptor-positive (ERþ) metastatic BC (mBC) have demonstrated genomic evolution during metastatic progression, and following treatment, such as the enrichment of HER2 and ESR1 genomic alterations (GAs) [2,3]. Clonal evolution can arise due to independent primary lesions, expansion of subclonal populations, or acquisition of novel GAs. Genomic changes following therapy are exemplified by acquired activating ESR1 GAs that mediate aromatase inhibitor (AI) resistance [2][3][4].
Clonal evolution processes highlight the importance of profiling a contemporaneous sample that is representative of disease progression to guide further management. However, limitations in performing repeated prospective biopsies of metastatic lesions over the disease course for a patient can present challenges for clinical genomic analysis [5]. Liquid biopsy and sequencing of circulating tumor DNA (ctDNA) from blood could provide a complementary approach to tissue-based genomic testing for mBC.
In this study, we carried out hybrid capture-based genomic profiling to characterize GAs in ctDNA from 254 patients with ERþ BC during the course of their clinical care.

Methods
Detailed descriptions of patient samples/methods are presented in supple mentary methods, available at Annals of Oncology online. Briefly, peripheral blood samples were collected from 254 patients with ERþ BC, plasma was isolated from 20 ml whole blood, !20 ng DNA was extracted, and hybrid capture-based genomic profiling of ctDNA was carried out in a CLIA-certified/CAP-accredited laboratory [Foundation Medicine (FM)] to identify substitutions, short insertions/deletions, rearrangements/fusions, and amplifications [12]. Sixty-two genes (supplementary Table S1, available at Annals of Oncology online) were sequenced (Illumina HiSeq 2500 or 4000) to a median unique coverage depth of 7503Â. Maximum somatic allele frequency (MSAF) was used to estimate the ctDNA fraction in plasma.

Patient characteristics
This study of hybrid capture-based sequencing of ctDNA in blood included consecutive genomic profiling results from 254 female patients with an initial diagnosis of ERþ BC, determined by routine IHC. Patient characteristics are described in Table 1  and supplementary Table S2, available at Annals of Oncology online. For patients with available clinical information, 94% were stage IV, 88% had received prior chemotherapy, and 88% had received prior AI in the adjuvant and/or metastatic setting.

Comparison of GAs in ctDNA and tissue
We compared frequently altered genes in ctDNA with ERþ BC tissue samples from the FM database and published studies [1][2][3] ( Figure 1C). For the majority of genes, mutation frequencies in ctDNA were similar to the range observed in tissue; ESR1 was mutated at a higher frequency (greater than twofold) compared with tissue, as expected from our study population comprising mostly patients who had received or were receiving AI treatment (supplementary Table S2, available at Annals of Oncology online) [9]. Amplifications were observed at lower frequencies in ctDNA compared with tissue, consistent with other studies of amplification detection in ctDNA from BC [7,13]. The estimated ctDNA fraction was significantly higher for cases with an amplification detected compared with cases without (supplementary Figure  S2B, available at Annals of Oncology online).
Genomic profiles of matched blood and tissue samples collected within 60 days of each other were available for 14 cases. We compared GAs assessed in both ctDNA and tissue ( Figure 1D; sup plementary Table S3, available at Annals of Oncology online). For short variant mutations, 89% (17/19) that were detected in tissue were also detected in ctDNA. Six mutations were detected in ctDNA only and two mutations were in tissue only. One ctDNA only ESR1 mutation (patient-5) was found in a second tissue sample from a distinct metastatic site, collected 408 days before blood sampling. One case (patient-10) harbored one shared and one tissue only ESR1 mutation; the allele frequency (AF) for the shared mutation (AF ¼ 34%) was 10-fold higher compared with the tissue only mutation (AF ¼ 3%). Two cases harbored both shared and ctDNA only mutations for the same gene (patient-8: ESR1; patient-12: PIK3CA): the shared mutation had a higher AF than the ctDNA only mutations in both cases (ESR1: twofold; PIK3CA: threefold), suggesting that ctDNA only mutations occur in less represented clones that may not be detected in a single tumor biopsy, consistent with clonal heterogeneity. For amplifications, 27% (3/11) that were detected in tissue were also detected in ctDNA; no amplifications were detected in ctDNA only. The estimated ctDNA fraction was higher for two cases where at least one amplification was detected in both tissue and ctDNA than for cases where amplification was detected in tissue only.

Landscape of ESR1 alterations in ctDNA
A total of 131 ESR1 GA were observed in 80 ERþ cases (supple mentary Figure S3A, available at Annals of Oncology online), including both ERþ/HER2À and ERþ/HER2þ cases ( Figure 1A and B); whereas, only 1 ESR1 GA (amplification) was observed in the ctDNA of 74 ER-cases (P ¼ 0.0001, Fisher's exact test, twotailed, supplementary Figure S3B, available at Annals of Oncology online). For the 130 ERþ patients with available clinical information regarding AI treatment, 35% (40/115) of all AI-treated patients had an ESR1 GA, and consistent with previous studies [9], ESR1 GAs were more frequent in patients treated with AI in the metastatic setting (40%, 36/91) versus patients treated with adjuvant AI only (11%, 2/19); all patients (40/40) with ESR1 GA had received prior AI (supplementary Table S2, available at Annals of Oncology online).
Multiple ESR1 GAs in the same sample are thought to be polyclonal in origin [9,10]. We carried out a pairwise assessment of all co-occurring ESR1 mutations to determine whether any      mutation pairs existed as compound mutations on the same allele (supplementary Table S4, available at Annals of Oncology online). Out of 67 mutation pairs, 49 were close enough to be evaluated on the same sequencing read; compound mutations were observed in 2/49 mutation pairs ( Figure 2C). In case-1, ESR1 L536F/D538G were observed as compound mutations on all reads. In case-2, ESR1 Y537N occurred as a single mutation in most reads, but a subset of reads harbored a compound mutation at the Y537 codon that resulted in conversion of Y537N to Y537S; the existence of two subsets of reads suggests sequential mutational events.
In addition to the GAs described above, 21 ESR1 variant of unknown significance (VUS) mutations were identified ( Figure 2D) in 15 ERþ cases including 5 cases with no ESR1 GAs (supplemen tary Figure S3A, available at Annals of Oncology online); no ESR1 VUS was observed in 74 ER-cases. To evaluate compound mutations, we analyzed 51 co-occurring ESR1 mutation pairs that involved an ESR1 VUS, and 20 could be evaluated on the same sequencing read. Compound mutations were observed in 4/20 mutation pairs and 2 mutation pairs existed as compound mutations in only a subset of sequencing reads (supplementary Figure  S5, available at Annals of Oncology online).
ESR1 rearrangements were observed in three cases: two ESR1 rearrangements had potential 3' fusion partners (AKAP12, NKAIN2) and one ESR1 rearrangement was fused to an intergenic region ( Figure 2E). ESR1-AKAP12 is recurrent in BC and all three ESR1 rearrangements resulted in loss of the ligandbinding domain (LBD), which likely results in constitutive ER activation [14,15]. Each ESR1-rearranged case harbored concurrent ESR1 mutation, suggesting prior AI exposure: we confirmed prior adjuvant AI and fulvestrant treatment of the patient with ESR1 fused to intergenic space.

Discussion
Genomic profiling of ctDNA has the potential to capture GAs that drive recurrent disease or therapeutic resistance and may provide an alternative when tissue biopsy is challenging. However, genomic profiles of ctDNA from ERþ mBC have not been extensively studied. We describe GAs identified in ctDNA from the blood of 254 patients with ERþ mBC.
Eighty-four percent of samples had evidence of ctDNA in the blood, consistent with a study of ctDNA release in mBC [8]. For cases with no evidence of ctDNA in the blood, lack of detectable somatic alterations is, in part, likely associated with insufficient ctDNA release into the blood at the time point of sampling that can be affected by clinical parameters including disease stage, tumor size, number of metastatic sites, albumin level, and number of lines of treatment [8,16]; these parameters were variable in this study of unselected cases (supplementary Table S2, available at Annals of Oncology online).
Alterations were identified in genes that have been associated with responses to targeted therapy in ERþ BC (PIK3CA, ESR1, ERBB2, FGFR1, BRCA1/2, AKT1) [4]. Compared with genomic studies of ERþ BC tissue biopsies, we identified similar mutation frequencies in ctDNA [1][2][3]. Tumor burden can be monitored by longitudinal assessment of variant AFs in ctDNA [8]. However, genomic profiling of large numbers of genes is best-suited for guiding therapy selection, but may be cost-prohibitive for serial testing. Instead, genomic profiling could establish GAs present in ctDNA at baseline for a patient, and guide design of personalized serial monitoring assays. GAs reported here could inform prioritization of genes to include for limited sequencing panels for longitudinal disease monitoring of ERþ mBC.
For a smaller subset of patients with temporally matched ctDNA and tissue samples, 89% of short variant mutations that were detected in tissue were also detected in ctDNA. Additional ESR1, TP53, and PIK3CA mutations were identified only in ctDNA; other studies have similarly observed additional mutations for each of these genes in ctDNA compared with matched tissue [5,10,17]. Additional mutations in ctDNA could reflect the utility of liquid biopsy to capture heterogeneity of metastatic sites in ERþ mBC [6]. Consistent with this idea, for paired cases with both shared and ctDNA only mutations in one gene, the shared mutation AF was two to threefold higher than the ctDNA only mutation AF. This hypothesis warrants confirmation in prospective trials and may be more relevant in clinical settings where targeted therapies are routinely employed.
In this study, genomic profiling was carried out as part of routine clinical care for unselected patients, including patients with low tumor burden; therefore, many samples had a low estimated ctDNA fraction (supplementary Table S2, available at Annals of Oncology online). The sensitivity for amplification detection in ctDNA was 27% for the 14 matched ctDNA-tissue pairs. Amplifications (including CCND1, FGFR1, ERBB2) were detected in ctDNA at lower frequencies than tissue; specifically ERBB2 amplification was identified in 29% of HER2þ cases. Detection of amplifications was associated with higher estimated ctDNA fraction. Our findings are consistent with NGS studies [using NGS panels or alternative approaches for amplification detection such as low coverage whole genome sequencing (plasma-Seq)] that highlighted the limitations for robust detection of amplifications in the context of low ctDNA fractions [18,19]. Other studies identified similarly low frequencies of ERBB2 amplification (21%-32%) in ctDNA from HER2þ BC and detected other common BC amplifications (including CCND1, FGFR1) at significantly lower frequencies in ctDNA compared with matched tissue [7,13]. Therefore, although amplifications may be detected in a subset of cases with sufficient ctDNA fraction, tissue-based genomic testing may be a more reliable method of detection. In BC, ERBB2 amplification remains the only established clinically utilized copy number biomarker, but amplifications including FGFR1 and 11q13 are being evaluated as biomarkers in trials [4]; tissue-based testing may be the preferable method for treatment selection based on copy number biomarkers.
We observed a high frequency of ESR1 GAs that are associated with AI resistance, as expected for this patient population of mostly mBC with prior AI treatment [9]. The ESR1 mutation frequency reported here is consistent with studies of AI-treated, ERþ mBC that used ddPCR to assess selected ESR1 mutations in ctDNA, including frequencies reported in several phase 3 trials [4,[9][10][11].
We observed a similar distribution of ESR1 mutations compared with a study of common ESR1 mutations in ctDNA using ddPCR [10]. Consistent with other studies, we frequently observed cases harboring >1 ESR1 GA [9,10]. Multiple ESR1 mutations are thought to reflect convergent evolution of distinct clones during AI resistance [6,10]; for the few cases evaluated using dual mutationspecific ddPCR probes, different ESR1 mutations existed on separate alleles [17,20]. We confirmed that most ESR1 mutation pairs occur on distinct sequencing reads, likely reflecting polyclonal origin; however, we also identified cases with ESR1 compound mutations on the same allele. Studies of ESR1 have focused on single mutations; ESR1 compound mutations in cis warrant further study, and such mutations might display differential therapeutic sensitivities compared with characterized single mutations.
Diverse ESR1 alterations were observed, including rearrangements with break points resulting in loss of the LBD. Similar ESR1 rearrangements with variable 3' fusion partners have been described and are activating [14,15]. ESR1 rearrangements demonstrate preclinical resistance to AI and SERDs, therefore, detection of ESR1 rearrangements may be important for therapy selection [15]. ESR1 VUSs reported here could represent novel functional mutations that warrant characterization.
We identify co-occurring alterations with ESR1, which could represent alternative targets or rational targets for combination therapy with SERDs [4]. Some of these GAs have been successfully targeted in the context of co-occurring ESR1 GA: responses have been observed for patients with concurrent ESR1/AKT1 mutation (to AZD5363) [21], ESR1/PIK3CA mutation (to alpelisib) [22], and for a patient from this study with ESR1/BRCA2 mutation (to olaparib; Dr S. Blau, personal communication).
Concurrent ESR1/ERBB2 activating mutations occurred in ERþ/HER2À BC and were more frequently observed in ctDNA compared with tissue, suggesting that ERBB2 and ESR1 mutations may commonly reside on distinct clones that may not be detected in a single tissue biopsy; ESR1 mutations were also observed in 25% of ERþ/HER2þ BC. Combinations of SERDs with HER2-targeted therapy could be relevant for such cases.
Here, we demonstrate the clinical implementation of genomic profiling of ctDNA from patients with ERþ BC and identify clinically relevant GAs. Blood-based testing may provide an alternative or complementary approach to tissue-based genomic testing for patients with mBC.