A Simple Method to Assess Estrogen Receptor Gene (ESR1) Amplification in Paired Biopsies from Primary Tumor and Recurrence in Breast Cancer Patients Receiving Endocrine Therapy

Globally, Breast Cancer (BC) is the leading cause of cancer death among women.
About 75% of patients are diagnosed with hormone-dependent tumors and are set
to receive Endocrine Therapy (ET) targeting the estrogen receptor. Unfortunately, a
significant proportion of these patients develops ET resistance. Still controversial, studies
have proposed that Estrogen Receptor-Alpha Gene (ESR1) alterations may underlie ET
resistance. Here, we describe the use of a Chromogenic in Situ Hybridization (CISH)
assay for the assessment of ESR1 amplification in primary tumors and recurrences. This
assay could be a useful clinical tool with therapeutic implications for estrogen receptor
positive BC patients.


Introduction
Worldwide, Breast Cancer (BC) remains as the leading cause of cancer-related death for women [1]. Approximately a 75% of breast tumors stain positive for the Estrogen Receptor (ER+). Upon diagnosis, ER+ BC patients are candidates to Endocrine Therapy (ET). Indeed, positivity for ER in tumors is a predictor for ET responsiveness [2,3]. Also, tumoral ER expression has prognostic implications, ER+ BC patients that receive ET display better survival rates versus ER-counterparts [4][5][6]. Furthermore, in early stage ER+ BC patients ET reduces mortality by 50% [4]. However, almost 25% of these patients will eventually develop ET resistance.
Among metastatic ER+ BC patients, this % is much higher. Clinically, ET resistance is manifested as recurrent disease during or after adjuvant treatment [3]. For these patients, metastasis is the main cause of death [7]. Studies have postulated a variety of ET resis-DOI: 10.26717/BJSTR.2020.25.004188 tance mechanisms, however one of the best described to date is ER-alpha gene (ESR1) amplification [8]. In fact, ESR1 amplification has been described in both primary tumors and metastases [9][10][11].
Currently, the assessment of gene amplification is a standard procedure routinely performed in pathology laboratories.
For example, Human Epidermal Growth Factor Receptor Type-2 (HER2) gene amplification is routinely determined by In Situ Hybridization (ISH) using Formalin-Fixed Paraffin Embedded (FFPE) tissue from mammary neoplasms for BC diagnosis. Herein, we report ESR1 amplification in paired biopsies (primary tumor and recurrence) from BC patients that developed ET resistance. To the best of our knowledge, this is the first report on this clinically relevant method using paired samples from primary tumor and its recurrence.

Study Design and Ethics Approval
This study is an observational, retrospective patient series.

Patients, Samples and Clinical Data
Patients with ER+ BC at diagnosis who were treated with ET and recurred during or after finishing treatment were selected from our database. Stage IV patients were excluded. FFPE tissue of tumors were obtained from a biobank. Anonymized demographics and clinical data were obtained from institutional database.

Situ Hybridization
FFPE blocks were sectioned at 5 um. After deparaffinization and hydration, a commercial ZytoLight® SPEC ESR1/CEN 6 Dual Color Probe (cat# Z-2069-200; ZytoVision GmbH, Germany) was used, following manufacturer's instructions. After ISH procedure, samples were blindly evaluated by an expert pathologist. ESR1 amplification was defined as a ratio of ESR1 to centromere at ≥4.

Statistical Analysis
Descriptive statistical analysis was used. Parametric or nonparametric tests were used when appropriate. Also, Spearman or Pearson correlations were assessed. Statistical significance was set at p<0.05. For data analysis, STATA v.14 and GraphPad Prism v7.0. software were used.
Also, in 3 out of 7 cases (42.9%) we observed discrepancies in ESR1-amplification status between the primary tumor and the recurrence; Within this subset, 2 went from ESR1-amplificationto + (primary versus recurrence), and 1 recurrence lost the ESR1amplification observed in the primary tumor. Additionally, patients with recurrent disease and ESR1-amplification+ tended to be older (63 versus 51 years, respectively), displayed higher BMI index and ER expression. They also had shorter disease-free survival rates against patients that changed their status from ESR1-amplification+ to -status. Unfortunately, all these differences were not statistically significant.

Discussion
To the best of our knowledge, this is the first report on ESR1amplification by CISH on paired samples from primary tumors and metastases obtained from ER+ BC patients. As reported previously, our data suggest ESR1 amplification is more frequently observed in recurrent tumors. Previous studies in early ER+BC patients demonstrate ESR1 genomic alterations are rare, ranging from 2-5% [12][13][14], suggesting they are not related to carcinogenesis [15]. In contrast, ESR1 alterations are far more frequent among metastatic ER+ BCs, ranging from 20-55% [16,17] [19,20].
Previously, studies have demonstrated that the rate of ESR1 amplification in BC is about 20% in both primary tumors and metastases [9][10][11]. Therefore, ET resistance cannot be fully attributed to this phenomenon. However, the amplification of mutant ESR1 variants may play a role in ET resistance. Interestingly, we found that 3 out of 10 (42.9%) displayed ESR1 amplification in primary tumors and 4 out of 8 (50%) had it at recurrence. Evidently, our study has certain limitations including the number of cases analyzed, also we did not assess point mutations on ESR1. Future studies should confirm our findings and elucidate the role of ESR1 amplification on the development of ET resistance.