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 Table of Contents  
Year : 2017  |  Volume : 3  |  Issue : 2  |  Page : 46-52

The application of estrogen receptor-1 mutations' detection through circulating tumor dna in breast cancer

Department of Medical Oncology, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

Date of Submission07-Mar-2017
Date of Acceptance17-Mar-2017
Date of Web Publication27-Apr-2017

Correspondence Address:
Fei Ma
Department of Medical Oncology, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ctm.ctm_10_17

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Breast cancer is the most common cancer in women worldwide. Endocrine therapy is the cornerstone of treatment for patients with hormone receptor-positive advanced breast cancer. Unfortunately, although most patients initially respond to endocrine treatment, they will eventually acquire resistance to endocrine therapy. The mechanisms of endocrine resistance are complicated. In particular, the estrogen receptor-1 (ESR1) mutation has been recognized as an important topic in recent years. Mutation of ESR1 leads to complete aromatase inhibitor resistance and partial resistance to estrogen receptor agonists and antagonists. Therefore, during clinical treatment, it is of great importance to continuously monitor ESR1 mutations before and after endocrine therapy. Conventional tissue biopsies have unavoidable disadvantages, and therefore, the use of circulating tumor DNA (ctDNA) has become more prevalent because it is noninvasive and convenient, has excellent sensitivity, and can quickly assess the overall situation of the tumor. The current methods for detecting ctDNA ESR1 mutations mainly include droplet digital polymerase chain reaction and next-generation sequencing techniques. Based on their advantages and disadvantages, we can establish an initial ESR1 mutation monitoring system. However, developing robust methods to monitor ESR1 mutation, detecting endocrine drug resistance, and evaluating prognoses for guiding clinical treatment strategies require long-term exploration. In this review, we will summarize recent concepts and advancements regarding ESR1 mutation monitoring, ctDNA detection technology, and their application in endocrine therapy of breast cancer.

Keywords: Circulating tumor DNA, endocrine resistance, estrogen receptor-1, monitor, mutation

How to cite this article:
Liu B, Yang Y, Yi Z, Guan X, Ma F. The application of estrogen receptor-1 mutations' detection through circulating tumor dna in breast cancer. Cancer Transl Med 2017;3:46-52

How to cite this URL:
Liu B, Yang Y, Yi Z, Guan X, Ma F. The application of estrogen receptor-1 mutations' detection through circulating tumor dna in breast cancer. Cancer Transl Med [serial online] 2017 [cited 2018 Apr 24];3:46-52. Available from: http://www.cancertm.com/text.asp?2017/3/2/46/205309

  Introduction Top

Breast cancer is the most common cancer in women and one of the leading causes of death worldwide.[1] Estrogen receptor alpha (ERα), encoded by the estrogen receptor-1 (ESR1) gene, is expressed in approximately 70% of all breast cancers, and endocrine therapy represents a major treatment modality in ERα-positive cancers.[2],[3] There are four main types of endocrine therapy for breast cancer: (1) selective ER modulators (for example, tamoxifen), (2) selective ER downregulators (for example, fulvestrant), (3) aromatase inhibitors (AIs, for example, anastrozole, letrozole, exemestane), and (4) gonadotropin-releasing hormone agonists (for example, goserelin).[3],[4] These agents are approved for the first-line hormonal therapy and have contributed to reduced recurrence rates and improved survival rates of breast cancer.[2],[3],[4],[5],[6] Although most hormone receptor (HR)-positive patients can benefit from these drugs, during endocrine therapy, endocrine resistance is common and essentially inevitable in advanced disease stages.[7],[8],[9]

Endocrine resistance can be divided into two types, intrinsic resistance, where ER-positive cancers never adequately respond to endocrine treatment, and acquired resistance, which develops following an initial response.[9] The mechanisms of resistance include (a) abnormal structure and function of ER, such as ESR1 mutation or amplification; (b) growth factor signaling pathway activation or enhancement, for example, mutation or amplification of human epidermal growth factor 2 (HER2), epidermal growth factor receptor, fibroblast growth factor receptor or mutations of downstream signaling molecules (PIK3CA, PI3K, mTOR); (c) cell aging and apoptosis-related gene mutations, such as TP53 mutations; (d) abnormal cell cycle regulation mechanisms, such as CCND1 amplification; and (e) changes in the tumor microenvironment.[3],[9],[10],[11] In general, the most common reason for endocrine resistance is ESR1 mutation.[12]

  Estrogen Receptor-1 Mutation Top

ESR1 mutations were first identified in patient xenograft studies reported in the 1990s.[9],[13] ESR1 mutations are numerically enriched in luminal A, PIK3CA-mutated, progesterone receptor-positive tumors.[14] ESR1 anomalies mainly include ESR1 translocation (chromosomal translocation and gene rearrangements), ESR1 amplification, and more commonly, ESR1 mutations.[15] Currently, there is no clear evidence that amplification and rearrangement are associated with drug resistance.[6],[7],[16] Almost all breast cancers that are initially hormone-dependent acquire antiestrogen resistance after repeated endocrine therapies and eventually become hormone-independent.[7],[8]

The representative mutation is the ESR1 ligand binding domain (LBD) "hot spot" mutation that is confined to codons 537 and 538 in exon 8.[2] To date, there are twelve known hot spot mutations that have been described. Among these mutations, the most frequent are Y537S, Y537N, Y537C, and D538G, which cover more than 80% of ESR1 mutations and are clustered in the LBD. Other mutations, including L536Q, L536R, P535H, V534E, K303R, S463P, and E380Q, at other positions have also been described.[2],[5],[14],[15],[17] Forty percent of patients had more than one ESR1 mutation, and in rare cases, patients had as many as four or five detectable, low-frequency ESR1 mutations.[2],[3],[4],[12],[14],[17],[18],[19] However, there was no evidence that multiple mutations were associated with a worse prognosis.[14]

ESR1 LBD mutations were thought to be extremely rare (< 1%) or undetectable in primary breast cancers, but in recent years, due to the maturity of droplet digital polymerase chain reaction (ddPCR) technology, we found that the ESR1 mutation rate in primary breast cancer is higher than previously estimated.[17] According to various cutoff values for detection, hot spot mutations of ESR1 can be found in approximately 2.5%–7% of primary breast cancers.[8],[17],[20],[21] However, after at least one-line endocrine therapy, 10.5%–86% (6/7) of breast cancer patients have an LBD mutation in ESR1, especially after serial endocrine therapy that includes AIs.[2],[4],[7],[8],[9],[12],[13] The difference in ESR1 mutation rate before and after treatment supports the idea that ESR1 mutation is generally an acquired resistance that emerges after long-term treatment with endocrine therapy.[9],[13] The more lines of endocrine treatment the patient receives, the higher the mutation rate of ESR1.[2],[4],[7],[8],[9],[12],[13] There are many potential reasons for the wide range of ESR1 mutation rates, including (1) differences in selected cutoff values, (2) small sample sizes in many studies, (3) varying inclusion criteria (for example, different treatment regimens and treatment times), (4) heterogeneity of the included populations (age, sex, race), and (5) different biopsy locations (primary or metastasis focused).[3],[4],[13],[17]

Y537N, Y537C, and D538G mutations can effectively promote the ER transactivation function, increase tumor cell sensitivity to estrogen, and even lead to ligand-independent activity, which can explain some of the reasons leading to acquired drug resistance.[7],[8] However, breast cancer cells with ESR1 LBD mutations have higher growth and survival rates than nonmutated cells in endocrine therapy, so the minority of the tumor cells harboring ESR1 mutations could become the dominant clone in the cancer.[5],[12] Mutated ESR1 can lead to complete AI resistance and partial resistance to ER agonists and antagonists.[2],[7] Gyanchandani et al.[3] found that ESR1-mutant breast cancers have faster progression (hazard ratio = 0.31; 95% confidence interval: 0.05–0.95) and significantly shorter progression-free survival (PFS) (median 9.0 vs. 4.2 months).[3] Therefore, understanding how to detect ESR1 mutations better and faster is particularly meaningful for clinicians to determine whether to continue the original endocrine therapy or to change the treatment regimen.[2],[5]

  Monitoring the Estrogen Receptor-1 Mutation in Circulating Tumor Dna Top

To make better use of ESR1 mutation information in breast cancer surveillance and to guide endocrine therapy, many studies have highlighted the necessity to monitor the ESR1 genotype regularly throughout the course of the disease.[2],[3],[22],[23] However, there are many limitations of traditional tissue biopsy (1) tumor biopsies are inconvenient from a scheduling perspective, especially when the tumor site is too deep or not easily accessible; (2) tissue biopsies usually cannot reflect the overall condition of the tumor because of intratumoral or intertumoral heterogeneity; (3) repeatedly taking biopsies from tumor tissue carries the risk of clinical complication; and (4) preservation of traditional tissue specimens (e.g., formalin-fixed paraffin embedded) is challenging.[5],[13],[23]

Circulating blood biomarkers, such as circulating tumor cells (CTCs) and cell-free plasma tumor DNA, have been developed as promising methods to help overcome the limitations associated with tumor biopsies and are considered to be alternative sources of tumor material.[2],[5],[13] CTCs are cancer cells that are detected in the blood of cancer patients. Their detection, quantification, and characterization represent a new method for cancer detection.[24] The use of CTCs to monitor ESR1 mutational status precedes the use of circulating tumor DNA (ctDNA). However, the use of CTCs is limited due to difficulties associated with isolation and relatively low detection rates.[24],[25],[25],[27]

Cell-free DNA (cfDNA) is short fragment of nucleic acids shed into the bloodstream by cells undergoing apoptosis or necrosis. Specifically, ctDNA refers to a portion of cfDNAs which are derived from tumor cells and contain the same mutations and genetic changes as cancer.[25],[28] Compared to CTC, ctDNA analysis is currently widely used due to excellent sensitivity and fast results.[24],[25] The ability to detect and quantify tumor mutations has proven to be effective in tracking tumor dynamics in real time, as well as serving as a liquid biopsy that can be used for a variety of clinical and investigational applications not previously possible.[28] In the SoFEA and PALOMA3 studies,[29],[30] ESR1 mutation status in ctDNA was successfully analyzed in 99.4% and 100% of plasma samples, respectively, which indicates that ctDNA has high detection rates. Takeshita et al.[5] also found that during endocrine treatment, ctDNA ESR1 mutation rates fluctuate more easily than those in tumor tissue, indicating that ctDNA may be more responsive to treatment. Meanwhile, many articles have confirmed the high degree of correlation between tumor tissue DNA and ctDNA analysis (57%–97%),[2],[4],[12] as well as the consistency between ctDNA and biopsy results.[2],[4] Thus, it is worth developing ctDNA as a noninvasive method to quickly assess mutational profiles.[5],[31]

  Circulating Tumor Dna Detection Technology Top

In general, ctDNA analysis has the potential to revolutionize the detection and monitoring of tumors.[32] High analytical sensitivity and specialized equipment are required for the detection of ctDNA because the quantity and quality of tumor-derived DNA can vary dramatically.[23] The current detection method for ctDNA ESR1 mutations mainly includes ddPCR and next-generation sequencing (NGS) techniques.[33],[36],[35] Guttery et al.[15] found that the NGS and ddPCR techniques have high detection accuracies for ESR1 mutations and are highly consistent with each other. Guttery et al.[15] confirmed that patients with ESR1 mutations were also prone to have PIK3CA, GATA3, and other mutations.[8],[14],[31]

The NGS and ddPCR techniques each have both advantages and disadvantages. NGS provides a relative panorama of the mutational landscape, including analyses of point mutations, amplifications, deletions, gene fusions, and translocations.[21],[36] NGS makes it feasible to discover novel chromosomal rearrangements and microbial infections and to resolve copy number alterations at a very high resolution.[36],[37],[38],[39],[40] With the help of NGS, we can understand the genomic complexity and the extensive intratumor genetic heterogeneity, which can lead to more precise treatment and management of individuals. Therefore, NGS has a great advantage, particularly in the exploration of unknown genetic variations.[13],[21] Although this technology has the capacity to generate the DNA sequence of hundreds of billions of nucleotides in a single experiment, the 1% error rate results in hundreds of millions of sequencing mistakes.[41] In theory, DNA subpopulations of any size should be detectable when deep sequencing a sufficient number of molecules, but the error rate limits the depth of the sequence,[41] and because of this, the biggest disadvantage of NGS is its relatively low sensitivity as compared to ddPCR. To overcome this disadvantage, new improvements in NGS based on safe-sequencing system and double-stranded DNA sequencing technology have appeared, dropping the error rate to every 10 nucleotides by eliminating DNA damage factors during sequencing.[41],[42],[43] However, even with these newly developed methods or reduced cutoff values,[4],[5],[12] the sensitivity of NGS is still lower than ddPCR. Therefore, many studies agree that ddPCR would be a more sensitive platform for ESR1 mutation detection.[4]

Common PCR techniques include real-time PCR (RT-PCR), 3D digital PCR (3dPCR), and ddPCR. The digital PCR concept has many potential advantages over RT-PCR, including the capability to obtain absolute quantification without external references and robustness to variations in PCR efficiency.[43] The positive rates detected by RT-PCR in cfDNA were significantly lower than those for NGS. However, the positive rates of cfDNA detected by ddPCR were higher than those for NGS in biopsy specimens.[4],[12],[43],[44] It is believed that the sensitivity of 3dPCR and ddPCR is similar.[43],[44],[45] Rather than in multi-well plates, ddPCR is a method that is based on reactions that occur in nanoliter-sized, aqueous droplets in oil. Rapid microfluidic analysis of thousands of droplets per sample makes ddPCR a high throughput method.[43] 3dPCR is a chip-based dPCR that has better stability and lower cost than the oil drop technology but has a lower detection speed and smaller throughput comparing to ddPCR.[43],[44],[45]

To overcome the low sensitivity of NGS, third-generation sequencing (TGS) was introduced.[46] TGS is designed to increase sequencing rates, throughput, and read lengths, to lower the complexity of sample preparation, and to ultimately decrease the cost of sequencing.[46] Single-molecule sequencing technologies, as a TGS method, do not use PCR amplification, thereby overcoming bias issues introduced by PCR amplification and dephasing.[46]

  Longitudinal Monitoring of Estrogen Receptor-1 Mutation in Clinical Practice Top

Crowley et al.[23] proposed that patients who become resistant to a particular therapy should undergo a tissue biopsy and that molecular tools (including ctDNA) would be useful.[47] Almost all articles agree that it is essential to monitor ESR1 mutation in ctDNA regularly throughout the entire course of the disease.[2],[3],[4],[5],[8],[12],[13],[15],[48],[49],[50],[51]

ESR1 mutations appear to evolve, and the ratio of ctDNA changes during treatment. The mutation rates of ESR1 increase gradually along with ongoing endocrine therapy.[2],[4],[7]-9, 12, 13 High mutation rates in ctDNA following therapy indicate heavy tumor burden, poor response to treatment, and short survival time.[2],[5],[13] During long-term monitoring, earlier ESR1 mutations are generally associated with worse prognoses.[3]

Dawson et al.[51] found that ctDNA levels showed a dynamic range and greater correlation with changes in the tumor burden. Spoerke et al.[14] also reported that patients with radiographic responses (complete and partial response) uniformly showed decreases in ESR1. Although the absolute quantity of ESR1 mutation can reflect the tumor burden and endocrine resistance in breast cancer, the absolute count of ctDNA is not the best indicator for disease. The quantity of ctDNA in the plasma of metastatic breast cancer was significantly higher than that of primary breast cancer, which has been confirmed by some experiments.[5] However, after surgery or medical treatment, the quantity of ctDNA in patients is reduced.[5] Therefore, Takeshita et al.[5] believed that the allelic ratios of mutant to wild-type ESR1 was a more precise biomarker than the absolute quantification or frequency of ESR1 mutations.

Mutational profiles can vary between different sites of metastatic disease.[4] Wang et al.[17] found higher allele frequency (34.3%–44.9%) in brain metastases than in bone metastases (1.4%), suggesting that the ESR1 mutation may be a driver of metastatic progression to this site. Therefore, if an ESR1-positive result is found during the monitoring process, clinicians should be aware of the risk of potential brain metastases in the future. However, in view of the small sample size of this study, this conclusion needs further confirmation.

Chu et al.[4] reported that ddPCR of ctDNA is capable of detecting ESR1 mutations even in patients without radiographic evidence of disease, which indicates that using ctDNA may avoid omissions of ESR1 mutation in micro-metastases. Some studies also report that using ctDNA for detection can help identify treatment for resistance up to 10 months before radiological methods.[52],[53]

Gu et al.[13] and Sefrioui et al.[2] both proposed a monitoring system proposing that baseline biopsies could be analyzed using NGS to yield a whole profile of relevant mutations in breast cancer including ESR1, PIK3CA, p53, and PTEN. After treatment, residual tumor biopsies and liquid biopsies can be collected and analyzed using ddPCR to confirm preexisting mutations and compare mutation frequency with matched baseline patient samples.

  Limitations and Challenges of Estrogen Receptor-1 Mutation Monitoring in Circulating Tumor Dna Top

First, most present published studies were retrospective, single-institute study, and small sample size reduces the credibility of the results. Second, relatively high costs may be a big problem limiting the clinical use of ESR1 mutation monitoring methods.[52],[53] Third, the standardization of ctDNA assays such as isolation technologies, standards, assay conditions, and more specifically, the time from blood draw to spinning, freezing plasma, and then thawing may also affect the variability of the data.[49],[54] Fourth, almost all the studies that used ddPCR as a detection method only queried for the most common ESR1 LBD mutations, such as D538G, Y537N, and Y537S, so it is likely that less frequent mutations associated with endocrine therapy resistance are missing.[4] Finally, Sefrioui et al.[2] reported that the mutated allelic fraction is generally lower in ctDNA than in metastatic biopsies, which may mislead clinical decision-making. A possible reason is that since circulating DNA includes DNA from tumoral ctDNA and nontumoral ctDNA, the nontumoral contingent may dilute the mutation signal.[2]

  Conclusion and Future Prospects Top

We currently know that ESR1 mutation is an important cause of endocrine drug resistance, but we do not know whether different ESR1 mutations have different roles in endocrine resistance. Toy et al.[8] showed that the ligand-independent activity of Y537S is stronger than that of D538G. The BOLERO-2 trial (NCT00863655) also found that everolimus prolonged PFS only in patients with D538G mutation, but not in patients with Y537S mutation.[55] Therefore, we hypothesize that different LBD mutations have different effects on endocrine resistance. Investigating whether ESR1 mutations can predict sensitivity to specific hormone therapies and searching for further correlation between different mutations of ESR1 and endocrine drugs are important next steps.

Another very realistic clinical endeavor is determining the treatment strategy for ESR1 positive or endocrine resistance patients. It is still being investigated if ERα LBD mutations retain sensitivity to other endocrine agents. It is currently uncertain what the most effective treatment for ESR1 mutant breast cancer is.[56] Clinical trials have confirmed that patients with ESR1 mutations had a poor PFS with subsequent AI-based therapies.[29],[56] Fulvestrant is an agent that has clearly demonstrated activity and good tolerability in women who have experienced disease progression on third-generation AIs.[57]In vitro studies and a Study of Faslodex with or without concomitant Arimidex versus Exemestane following progression on Nonsteroidal Aromatase inhibitors (SoFEA) have both suggested that mutant ERα leads to relative resistance to exemestane and relative sensitivity to fulvestrant, but higher doses are required to inhibit mutant ERα.[14],[29],[56] In CONFIRM trial, 500 mg of fulvestrant had significantly prolonged PFS better than 250 mg of fulvestrant and was not associated with increased toxicity.[58] The Phase III FALCON study (NCT01602380) also found 500 mg of fulvestrant compared with 1 mg of anastrozole can significantly prolong PFS.[59] Targeting of signaling pathways during endocrine resistance has been shown to be effective, as demonstrated by improvement in PFS. At least two studies, including the BOLERO-2 trial, have confirmed that the addition of everolimus (an mTOR inhibitor) to endocrine therapy resulted in improved clinical outcomes.[60],[61] The palbociclib combined with fulvestrant in HR + HER2-negative metastatic breast cancer after endocrine failure (PALOMA-3) study also demonstrated that palbociclib (cyclins CDK4/6 inhibitor) improves PFS when combined with 500 mg of fulvestrant in patients with progression after receiving prior endocrine therapy.[56],[62] Another CDK4/6 inhibitor ribociclib is also proved to improve PFS when combined with letrozole in MONALEESA-2 trial.[63]

In summary, according to the current study, the alternative options to treat endocrine resistance include the following: (1) using tamoxifen or fulvestrant for patients with acquired resistance after AIs;[57] (2) increasing the dose of tamoxifen or fulvestrant;[58] (3) using inhibitors for growth factor pathways (e.g. mTOR inhibitors, CD4/6 inhibitors);[60],[61],[62] and (4) using stronger or novel antiestrogen drugs.[13],[57],[58] Further clinical research is needed to determine if these methods can be truly beneficial. Before using ddPCR for detecting ESR1 mutations to guide therapy being adopted into clinical practice, many issues require careful examination. It is unknown what allelic frequency of ESR1 mutation is associated with symptomatic disease progression and whether changing endocrine therapies can improve patient outcomes.[4]

Although TGS technology is currently popular, its use in ESR1 mutation detection has not yet been confirmed, and therefore, its potential feasibility, sensitivity, and accuracy are currently unknown. With further application of TGS technology, we can better understand the breadth and sensitivity of drug-resistant mutation detection.

At present, the studies based on ESR1 detection in ctDNA are similar and do not offer any innovative ideas or novel suggestions. In summary, longitudinal detection of ESR1 mutations plays an integral role in predicting the efficacy of endocrine therapy, monitoring endocrine therapy resistance, and evaluating prognosis.[2] Therefore, the application value of ctDNA to breast cancer is self-evident. With improved NGS and ddPCR detection technologies, we can better monitor ESR1 mutations. However, promoting a more reasonable ESR1 mutation monitoring system and using ctDNA to achieve accurate endocrine therapy will be a long-term task.

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Conflicts of interest

There are no conflicts of interest.

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