Navigation through inter- and intratumoral heterogeneity of endocrine resistance mechanisms in breast cancer: A potential role for Liquid Biopsies?

Abstract

The majority of breast cancers are hormone receptor positive due to the expression of the estrogen and/or progesterone receptors. Endocrine therapy is a major treatment option for all disease stages of hormone receptor–positive breast cancer and improves overall survival. However, endocrine therapy is limited by de novo and acquired resistance. Several factors have been proposed for endocrine therapy failures, which include molecular alterations in the estrogen receptor pathway, altered expression of cell-cycle regulators, autophagy, and epithelial-to-mesenchymal transition as a consequence of tumor progression and selection pressure. It is essential to reveal and monitor intra- and intertumoral alterations in breast cancer to allow optimal therapy outcome. Endocrine therapy navigation by molecular profiling of tissue biopsies is the current gold standard but limited in many reasons. “Liquid biopsies” such as circulating-tumor cells and circulating-tumor DNA offer hope to fill that gap in allowing non-invasive serial assessment of biomarkers predicting success of endocrine therapy regimen. In this context, this review will provide an overview on inter- and intratumoral heterogeneity of endocrine resistance mechanisms and discuss the potential role of “liquid biopsies” as navigators to personalize treatment methods and prevent endocrine treatment resistance in breast cancer.

Keywords

Breast cancer endocrine resistance intratumoral heterogeneity intertumoral heterogeneity liquid biopsy

Introduction

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among women worldwide, accounting for 25% of the total cancer cases (1.68 million) and 15% of the cancer deaths (520,000).^1,2 Approximately 70% of advanced breast cancers are hormone receptor positive (HRpos). HRpos breast cancers are defined either by expression of estrogen receptor (ER) or progesterone receptor (PR) or both.^3,4 The HR status is assessed by immunohistochemical analyses of formalin-fixed and paraffin-embedded sections of tumor tissue and application of a scoring system.⁵ According to the current guidelines defined by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP), a tumor is considered as ER or PR positive if there are at least 1% of positively stained nuclei.⁶ HR expression levels influence sensitivity to endocrine therapies (ETs) like aromatase inhibitors (AIs), selective ER modulators (SERMs), or selective ER down regulators (SERDs). Clinical studies demonstrated the therapeutic efficacy of ET for women with HRpos primary tumors or metastatic disease.^7,8 However, refractory to first-line ET is observed in 20%–25% of patients.^9,10 For second or greater lines of therapy, the clinical benefit rate declines to about 30%.¹¹ Therefore, real-time monitoring of ET resistance mechanisms is of great purpose for clinicians to determine whether to proceed with current ET or to change the treatment regimen. Insights into current clinical approaches and challenges will be provided in the following paragraphs.

ET can be challenged by molecular, phenotypic, and functional diversity within a patient’s tumor. Recent advances in transcriptomic and genomic technologies have led to improvements and new insights in understanding heterogeneity in breast tumors¹² which comprises intra- and intertumoral heterogeneity in primary tumors, metastasis, circulating-tumor cells, as well as cell-free DNA (cfDNA)^13,14 and indicates that heterogeneity in breast cancer is a main driver to develop resistance to ET.¹⁵ Assessment of heterogeneity in breast cancer is currently based on molecular and phenotypic analysis of tissue biopsies. Although tissue biopsies represent the gold standard in tumor profiling, tissue biopsies are not without adverse risk, provide only a snapshot in time and location and are limited in understanding tumor dynamics. The presence of intra- and intertumoral heterogeneity may also limit the tissue-based profiling, especially when mechanisms for ET resistance need to be evaluated in disease progression. In light of these limitations of tissue biopsies, “liquid biopsies” offer hope to overcome some of the limitations. In principle, “liquid biopsies” can provide the same genetic information as tissue biopsies. “Liquid biopsies” hold clear advantages in allowing repeatable non-invasive assessing of the clonal dynamics throughout therapy and early identification of therapeutic resistance driver.¹⁶

In this context, we will highlight inter- and intratumoral heterogeneity of ET resistance mechanisms and discuss the potential role of “liquid biopsies” as an additional clinical tool to personalize treatment methods and to prevent ET resistance in breast cancer.

Targeted ET and endocrine resistance in breast cancer

For over 30 years, targeted ET methods for estrogen receptor–positive (ERpos) breast tumors are available in clinics and have a significant impact on mortality.^17,18 Multiple pharmaceutical agents are effective in HRpos breast cancer. These include AIs (e.g. anastrozole, letrozole, and exemestane) and gonadotropin-releasing hormone agonists (GnRHa) that reduce estrogen biosynthesis, SERMs (e.g. tamoxifen, toremifene, or raloxifene), and SERDs (e.g. fulvestrant). In patients with HRpos breast cancer, these pharmaceutical drugs significantly improve survival.¹⁸ Approximately 30%–40% of HRpos tumor patients have an objective response rate to ET¹⁹ but less side effects compared to cytotoxic therapy.²⁰ Standard ET is 5 years but might be considered up to 10 years based on the individual risk of relapse.²¹ Duration, choice, and sequence of ET mainly rely on menopausal status and side effects of pharmaceutical agents. Contemporary adjuvant ET options for premenopausal women include tamoxifen with or without GnRHa/ovarian ablation (OA), an AI with GnRHa/OA, or GnRHa/OA alone.²² For postmenopausal women, contemporary ET regimes suggest treatment of an AI alone, tamoxifen alone, or sequential therapies consisting of an AI and tamoxifen.²³ Nevertheless, failure of ET for women with HRpos primary tumor or metastatic disease is observed in 20%–25% of patients.^10,24

Endocrine drug resistance can be separated into two basic ways of drug failure: primary or de novo and secondary or acquired. On one hand, ET to HRpos breast cancers does not elicit any response (de novo or primary resistance). On the other hand, breast tumors with a good initial response may progress sooner or later (acquired or secondary resistance).²⁵ Several studies in breast cancer proposed that drug resistance might be due to the tumor’s heterogeneous molecular alterations.²⁶ In recent years, several molecular alterations have been proposed to mediate endocrine resistance including altered ER expression, mutations in ER-alpha (ESR1), altered expression of growth factor receptors, activating phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling, altered expression of cell-cycle regulators, overexpression of ER coactivators, autophagy, and epithelial-to-mesenchymal transition (EMT).²⁷ Given the inherent heterogeneity of ERpos disease, current research aims to restore sensitivity to ET or to prevent resistance outright by early detecting and monitoring of tumor heterogeneity.

Intra- and intertumoral heterogeneity in breast cancer

Breast tumors are characteristically heterogeneous displayed by molecular, phenotypic, and functional diversity. To date, the origins of inter- and intratumoral heterogeneity are not fully understood.²⁸ Possible sources for cancer diversity are cell-autonomous, non-cell-autonomous, and stochastic events.²⁸ Intratumoral heterogeneity may be observed by existence of subclonal diversity within a tumor (e.g. primary tumor, metastasis, recurrence lesion, and circulating-tumor cells (CTCs)). Genetic and phenotypic alterations between primary tumors, metastasis, recurrence lesions, CTCs, and cfDNA describe intertumoral heterogeneity. Delineation of both forms of heterogeneity is essential to better understand therapy resistance and to define new routes for subsequent individualization of ETs (Figure 1).

Figure 1.

Theoretical model illustrating intra- and intertumoral heterogeneity in breast cancer: cell-autonomous, non-cell-autonomous (e.g. ET), and stochastic events generate subclonal diversity leading into intra- and intertumoral heterogeneity over time. Intertumoral heterogeneity may present by existence of molecular and phenotypic diversity between matched pairs of primary tumors, metastasis, recurrence lesions, circulating-tumor cells and cfDNA. Within a primary tumor, metastasis, recurrence lesion, or circulating-tumor cells, subclonal diversity may also be observed (intratumoral heterogeneity).

Assessment of tumor heterogeneity—tissue biopsies

Assessment of heterogeneity in breast cancer is currently based on molecular and phenotypic analyses of tissue biopsies. In the clinical setting, biopsies are obtained by core needle biopsy (CNB), incisional biopsy, or excisional biopsy (EB) for histopathological characterization. Although tumor tissue is the gold standard for clinical and investigational analyses, tissue biopsies are not without adverse risk, provide only a snapshot in time and location, and are limited in understanding tumor dynamics.

Assessment of tumor heterogeneity—liquid biopsies

To overcome these limitations, the idea of liquid biopsies was developed as a new way allowing non-invasive serial assessment of biomarkers which captures intra- and intertumoral heterogeneity. There are two main promising non-invasive types of liquid biopsies: CTCs and ctDNA.²⁹

CTCs

Circulating-tumor cells are deemed to be evading cancer cells that have been shed or actively invaded from the primary tumor into the blood circulation or lymph system and which may finally extravasate to found metastases. Therefore, they are considered—at least in part—to being metastatic progenitors. In the blood, CTCs are present at a ratio of approximately 1 CTC to 10⁶–10⁷ of peripheral blood cells.³⁰ Their presence is associated with worse clinical outcome and can be utilized to optimize personalized management of metastatic breast cancer. Recent studies revealed their strong clinical value.^31–33

CtDNA

Circulating-tumor DNA is released from apoptotic or necrotic tumor cells.³⁴ It is a fraction of the total pool of cfDNA. CtDNA is normally measured in plasma or serum but has also been detected in other blood fractions such as extracellular vesicle exosomes,³⁵ platelets,³⁶ and buffy coat.³⁷ In line with the presence of CTCs, the presence of ctDNA was shown to correlate with tumor burden in breast cancer.³⁸

Tumor heterogeneity and ET resistance

Heterogeneity enhances the robustness of tumors and therefore challenges cancer therapies.²⁸ Compared with monoclonal tumors, tumors exhibiting inter- and intratumoral heterogeneity are reacting with different sensitivities to therapeutic interventions. Studies indicate that tumor heterogeneity contributes to ET failure.²⁶ Therefore, it is essential to monitor and reveal intra- and intertumoral heterogeneity of endocrine resistance mechanisms to allow optimal therapy outcome. Intra- and intertumoral heterogeneity of endocrine resistance mechanisms are listed in Table 1.

Table 1.

Intra- and intertumoral heterogeneity of endocrine resistance markers.

Heterogeneity	Matched pairs	Marker	Alteration	Matched pairs, SG (D)	Appearance	Therapy	Ref.
ESR1
Inter	P–M	ESR1	Mutation	P, M 13 (5)	5 G in M	ET	Merenbakh-Lamin et al.³⁹
Inter	P–M; P–R	ESR1	Mutation	P, M/R 8 (4)	3 G in M 1 G in R	ET, –	Takeshita et al.⁴⁰
Inter	P–M; P–ctDNA	ESR1	Mutation	P, M, ctDNA 1 (1)	1 G in M and ctDNA	ET	Bardia et al.⁴¹
Inter Intra	P–C; C–C; C–plDNA	ESR1	Mutation	P, C 4 (2) C 4 (2) P/C, plDNA 4 (0)	2 G in C 2 G	ET	Shaw et al.⁴²
Inter	P–C (in vitro)	ESR1	Mutation	PT, C 4 (1)	1 G in C	ET	Yu et al.⁴³
Inter	M–ctDNA	ESR1	Mutation	M, ctDNA 5 (2)	2 G in ctDNA	ET	Wang et al.⁴⁴
Inter	P–ctDNA; M–ctDNA	ESR1	Mutation	P, ctDNA 42 (40) M, ctDNA 47 (25)	40 G in ctDNA 4 L in ctDNA 21 G in ctDNA	–, ET	Spoerke et al.⁴⁵
Intra	P–P	ESR1	Mutation	PT 38 (2)	2 G	ET^a	Miller et al.⁴⁶
Growth Receptors/Factors
Intra	C–C (in vitro)	FGFR1	Amplification	C 10 (8)		NA	Khoo et al.⁴⁷
Inter	P–C (in vitro)	FGFR2	Mutation	P, C 4 (1)	1 G in C	ET	Yu et al.⁴³
Inter	P–R	HER2	Amplification/ Expression	P, R 42 (11)	11 G/L in R	ET	Drury et al.⁴⁸
Inter	P–R	HER2	Amplification/ Expression	P, R 26 (3)	3 G in R	ET	Gutierrez et al.⁴⁹
Inter	P–M	HER2	Expression	P, M 96 (16%)	NA	ET, CT	Aktas et al.⁵⁰
Inter	P–M	HER2	Expression	P, M 31 (17)	15 G in M 2 L in M	NA	Regitnig et al.^{51 b}
Inter	P–M	HER2	Expression	P, M 50 (5)	2 G in M 3 L in M	NA	Sun et al.⁵²
Inter	P–C; M–C	HER2	Expression	P, C 36 (59%/58%) M, C 36 (67%/53%)	NA	ET, CT	Aktas et al.⁵⁰
Inter	P–C	HER2	Amplification	P, C 88 (15%)	5 G in C 8 L in C	NA	Krishnamurthy et al.⁵³
Inter	P–C	HER2	Amplification/ Expression	P, C 40 (13)	8 G in C 5 L in C	NA	Pestrin et al.⁵⁴
Inter	P–C	HER2	Amplification	P, C 31 (3%)	4 L in C	NA	Meng et al.⁵⁵
Inter	P–C	HER2	Expression	P, C 37 (15) 21 (9)	10 G in C 5 L in C 3 G in C 6 L in C	–, CT	Riethdorf et al.⁵⁶
Inter Intra	P–C; C–C	HER2	Amplification	P, C 40 (8) C 42 (14%)	5 G in C 3 L in C	NA	Polzer et al.²⁶
Inter	P–plDNA	HER2	Amplification	P, plDNA 8 (4)	4 L in plDNA	NA	Shaw et al.⁵⁷
Intra	C–C (in vitro)	HER2	Amplification	C 10 (8)		NA	Khoo et al.⁴⁷
Intra	C–C	HER2	Amplification	C 24 (3)		–	Riethdorf et al.⁵⁶
Intra	C–C	HER2	Expression	C 33 (15)		–, CT, ET, AB	Gasch et al.⁵⁸
Inter	P–R	IGF1R	Expression	P, R 39 (75%)	29 L in R	ET	Drury et al.⁴⁸
Inter	P–P	IGF1R	Expression	P - P 63 (22)	11 G 11 L	–, ET, CT	Dhasarathy et al.⁵⁹
Inter	P–P; M–M	IGFR1	Expression	P 26 M 29	−0.62× change −0.55× change	ET	Arnedos et al.⁶⁰
Inter	P–M	VEGF	Expression	P, M 50 (29)	9 G in M 20 L in M	NA	Sun et al.⁵²
PI3K/AKT/mTOR/FOXO signaling
Inter	P–M	PI3K	Mutation	P, M 100 (31)	21 G in M 11 L in M	ET, CT	Dupont Jensen et al.⁶¹
Inter	P–M	PI3K	Mutation	P, M (50) 18%	4 G in M 5 L in M	ET, CT	Gonzalez-Angulo et al.⁶²
Inter Intra	P–C; C–C	PI3K	Mutation	P, C 6 (66%) C 16 (10)	3 G in C 1 L in C	NA	Polzer et al.²⁶
Inter	P–C (in vitro)	PI3K	Mutation	P, C 4 (1)	1 G in C	ET	Yu et al.⁴³
Inter Intra	P–C; C–C	PI3K	Mutation	P, C 6 (1) C 18 (2)	1 G in C	NA	Pestrinet al.⁶³
Inter	PT/M–ctDNA	PI3K	Mutation	PT/M, ctDNA 142 (14%)	5 G in ctDNA 14 L in ctDNA	–, ET	Spoerke et al.⁴⁵
Inter	P/M–cfDNA	PI3K	Mutation	P/M, plDNA 41 (3/4)	2/4 L in cfDNA 1/0 G in cfDNA	NA	Board et al.⁶⁴
Intra	P–C; C–C; C–plDNA	PI3K	Mutation	P, C 4 (0) C 4 (1) P/C, plDNA 4 (0)	1 L	ET	Shaw et al.⁴²
Intra	C–C	PI3K	Mutation	C 33 (9)		CT, ET, AB	Gasch et al.⁵⁸
Intra	C–C	PI3K	Mutation	C 12 (1)		NA	Neves et al.⁶⁵
Intra	P–P	PI3K	Mutation	P 10 (4)		ET, CT	Dupont Jensen et al.⁶¹
Intra	C–C	AKT, mTOR, FOXO	Expression	C 2 (2)		NA	Gorges et al.⁶⁶
Inter	P–R	PTEN	Expression	P, R 45 (8)	3 G in R 5 L in R	ET	Drury et al.⁴⁸
Inter	P–M	PTEN	Expression	P, M 51 (26%)	8 G in M 5 L in M	ET, CT	Gonzalez-Angulo et al.⁶²
Inter	P–M	PTEN	Mutation	P, M 33 (1)	1 G in M	CT, ET, AB	Meric-Bernstam et al.⁶⁷
Cell cycle
Inter	P–M	CCND1	Amplification	P, M 33 (1)	1 G in M	CT, ET, AB	Meric-Bernstam et al.⁶⁷
Inter	P–M	Rb	Mutation	P, M 33 (2)	2 G in M	CT, ET, AB	Meric-Bernstam et al.⁶⁷
Inter	P–M	CDK4	Amplification	P, M 33 (4)	4 G in M	CT, ET, AB	Meric-Bernstam et al.⁶⁷
Intra	C–C	CCND1	Amplification	C 10 (8)		NA	Khoo et al.⁴⁷
Autophagy
Inter	P–M (in vivo)	GRP78	Expression	P, M 3 (3)	3 G in M	–	Miao et al.⁶⁸
Inter	P–M	GRP78	Expression	P, M 27 (8)	3 G in M 5 L in M	NA	Bartkowiak et al.⁶⁹
EMT
Intra	C–C	VIM	Expression	C 5 (5)		–	Polioudaki et al.⁷⁰
Intra	C–C	VIM	Expression	C 22 (19)		NA	Bourcy et al.⁷¹
Intra	C–C	VIM	Expression	C 50 (44)		NA	Kallergi et al.⁷²
Intra	C–C	TWIST1	Expression	C 50 (47)		NA	Kallergi et al.⁷²
Intra	C–C	CDH2	Expression	C 6 (4)		–	Armstrong et al.⁷³

SG: study group; D: discordance; L: loss; G: gain; Inter: intertumoral; Intra: intratumoral; P: primary tumor; M: metastasis; C: circulating-tumor cells; ctDNA: circulating-tumor DNA; plDNA: plasma DNA; cfDNA: cell-free DNA; ET: endocrine therapy; CT: chemotherapy; AT: antibody therapy; (–): no medical therapy; NA: not available; ESR1: estrogen receptor alpha gen; FGFR1: fibroblast growth factor receptor 1; FGFR2: fibroblast growth factor receptor 2; IGF1R: insulin-like growth factor 1 receptor; HER2: human epidermal growth factor receptor 2; VEGF: vascular endothelial growth factor; PI3K: phosphoinositide-3-kinase; AKT: protein kinase B; mTOR: mechanistic target of rapamycin; FOXO: forkhead box proteins; PTEN: phosphatase and tensin homolog; CCND1: cyclin D1 gene; Rb: retinoblastoma tumor suppressor protein; CDK4: cyclin-dependent kinase 4; GRP78: 78 kDa glucose-regulated protein; VIM: vimentin; TWIST1: twist-related protein 1; CDH2: N-cadherin.

Intra- and intertumoral heterogeneity of mutations in ESR1, growth factor receptor signaling, PI3K/Akt/mTOR signaling, expression of cell-cycle regulators, autophagy and epithelial-to-mesenchymal transition (EMT) are evident in breast cancer.

Gained through AI treatment.

Overview of HER2 discordance in matched primary tumor and metastasis.

ER expression

Endocrine resistance due to heterogenic expression of ER is the focus of current studies. Intertumoral heterogeneity of ER and PR expression could be shown between primary tumor, corresponding metastasis, and CTCs. In several studies, ER and PR were differently expressed in primary tumor and corresponding metastasis.^50,74–79 Therefore, the international and national guidelines of the AGO Breast currently recommend the reassessment of the HR status in the metastatic setting. Paoletti et al.⁸⁰ examined ER expression on CTCs in patients having ERpos primary tumors. In all, 13 patients with ERpos primary tumors were diagnosed with ERneg CTCs.⁸⁰ Aktas et al.⁵⁰ also observed intertumoral heterogeneity between ER expression in primary tumors and matched CTCs. Moreover, no correlation was found between the ER staining intensity in the primary tumor and corresponding CTCs.¹⁴

Intratumoral heterogeneity of ER expression could be shown within primary tumors, within metastasis, and CTCs. Collin et al.⁸¹ observed intratumoral heterogeneity of ER expression in primary breast cancers. Paoletti et al.⁸⁰ identified intratumoral heterogeneity of ER expression levels within 790 CTCs of one patient. Babayan et al.¹⁴ demonstrated CTC patient samples with either homogenously ERpos (5/16) or ERneg (3/16) CTCs but also samples with ER heterogeneity (8/16).

To sum up, Bouckaert et al.⁸² reported about correlation between ER expression and rate of response to ETs. Nevertheless, in most cases of acquired endocrine resistance, expression of ER is retained.⁸³ Ellis et al.⁸⁴ showed in ERpos breast cancers at relapse a loss of ER expression in only 20% of cases. If loss of ER expression is not the underlying mechanism explaining endocrine resistance in these patients, other main drivers like ESR1 mutations may be the reason.⁸³

ESR1 mutations

Nearly two decades ago, the first mutations in the ERS1 gene coding for ER were reported in patient xenograft models.^85,86 Recently, next-generation sequencing (NGS) studies revealed that hot spot mutations in the ESR1 gene locus act as drivers for ET resistance.^87,88 Several ESR1 mutations associated with acquired resistance to antiestrogen therapy were reported in the last decade.^87,88 They seem to be rare in native primary breast cancer tumors⁸⁹ but can be observed more frequently in treated ERpos breast cancer and metastases.

Intertumoral heterogeneity of ESR1 mutations was determined between primary tumors, metastatic lesions, CTCs, and cfDNA. They were found in metastases after ET which were absent in primary tumors.³⁹ In line, Takeshita et al.⁴⁰ detected three patients with ESR1 mutations in metastatic lesions and one ESR1 mutation in recurrence which were absent in the ERpos primary tumor. Interestingly, two ESR1 mutations appeared without any ET.⁴⁰ Bardia et al.⁴¹ reported about a 67-year-old woman with ERpos breast cancer metastasis resistant to letrozol. Sequencing of the metastatic lesion revealed an ESR1 mutation which was absent in the primary tumor. Moreover, the same ESR1 mutation could also be detected in ctDNA extracted from the blood sample.⁴¹ In line, intertumoral heterogeneity of ESR1 mutations between primary tumor and CTCs could be shown by Shaw et al.⁴² ESR1 mutations detected in CTCs were absent in primary tumor tissue of two patients. In vitro CTC cultures of matched primary tumors could also demonstrate intertumoral heterogeneity.⁴³ Intertumoral heterogeneity between primary tumor and ctDNA was demonstrated in a study of Wang et al.⁴⁴ In three patients, ESR1 mutations were detected in both metastatic tissue and ctDNA, whereas in two patients ESR1 mutations were only found in ctDNA.⁴⁴ In line, intertumoral heterogeneity between matched pairs of primary tissue and ctDNA or metastatic tissue and ctDNA could be also shown by Spoerke et al.⁴⁵

Intratumoral heterogeneity was found within primary tumors and CTCs. Miller et al.⁴⁶ showed intratumoral heterogeneity of ESR1 mutations within breast cancer tumors after treatment with AIs. Primary tumors demonstrated remodeling of the clonal architecture and formation of new ESR1 mutations as well as enrichment of ESR1 mutations.⁴⁶ Two patients with metastatic breast cancer revealed ESR1 heterogeneity between individual CTCs.⁴² Taken together, inter-and intratumoral heterogeneity of ESR1 mutations is present in breast tumors. They seem to be a rare cause of de novo endocrine resistance but more common in acquired resistance after ET.

Growth receptors/factors

Changes in expression, amplifications as well as mutations of growth factor receptors (e.g. fibroblast growth factor receptor (FGFR), human epidermal growth factor receptor (HER2), insulin-like growth factor (IGFR), vascular endothelial growth factor receptor (VEGFR), and epidermal growth factor receptor (EGFR)) might also contribute to loss of endocrine responsiveness. The bidirectional crosstalk between ER and growth factor receptors/pathways has been well documented.^90,91

FGFR

Approximately up to 9% of all breast cancer harbor FGFR1 (fibroblast growth factor receptor 1) amplification and 4% FGFR2 (fibroblast growth factor receptor 2) amplification.^89,92,93 In metastatic breast cancer, the FGFR1 gene is more often amplified than the FGFR2 gene.⁹⁴ Early relapse of ERpos tumors is associated with amplification of FGFR.⁹² Several studies have found correlations between FGFR1 amplification and its expression,^95–97 whereas others have not.^98,99 In 8 of 10 patient samples, intratumoral heterogeneity between FGFR1 copy number variants of CTCs was observed.⁴⁷ FGFR2 mutations were detected in cultured CTCs of one breast cancer patient which were absent in matched primary tumor.⁴³

HER2

The human epidermal growth factor receptor 2 (HER2) is overexpressed in approximately 10% of ERpos breast cancers.¹⁰⁰ In ERpos breast tumors, HER2 gene amplification and/or overexpression is associated with minor response to ET.^100–103

Intertumoral heterogeneity of HER2 could be shown between primary tumor, metastasis, and CTCs. Drury et al.⁴⁸ observed intertumoral heterogeneity of HER2 expression in 11 recurrent tamoxifen-resistant tumors compared to matched primary tumors. In line, Guttierez et al.⁴⁹ demonstrated a switch from 11% (3/26) HER2neg primary breast tumors to HER2 amplified and/or overexpressed ones at time of tamoxifen resistance. Discordance in HER2 positivity in 16% of primary tumors and metastasis was reported by Aktas et al.⁵⁰ In line, Regitnig et al.⁵¹ observed increased HER2 expression in 15 cases and decreased HER2 expression in 2 cases of distant metastasis compared to primary tumors. Sun et al.⁵² observed 50 samples of primary breast cancer and corresponding metastasis. Three HER2-positive primary breast cancers revealed a negative HER2 status in metastases, whereas two HER2 negative primary breast cancers showed positive metastases.⁵² Discrepancies between HER2 status in primary tumor and CTCs has been described in multiple studies.^50,56,55 In the study of Aktas et al., HER2 expression of CTCs was analyzed with two methods: AdnaTest and CellSearch^® assay. AdnaTest demonstrated a 59% concordance between primary tumor and CTCs. Between metastasis and CTCs, the concordance value was 67%. CellSearch^® revealed a concordance of 58% between primary tumor and CTCs as well as 53% between metastasis and CTCs.⁵⁰ In line, Krishnamurthy et al.⁵³ found a discordance in HER2 status between primary tumor and CTCs of approximately 15%. Also, Pesztrin at al. could demonstrate a non-concordant HER2 status in 32% of cases.⁵⁴ In another study, the HER2 status of primary tumor and corresponding CTCs was discordant in 3% of 31 cases.⁵⁵ The GeparQuattro trial revealed intertumoral heterogeneity of HER2 expression in matched CTCs and primary tumors before and after neoadjuvant Herceptin treatment.⁵⁶ Polzer et al.²⁶ observed loss and gain of HER2 amplification in eight matched primary tumors and CTCs. Concerning plasma DNA, in four of eight HER2-positive primary tumors, HER2 amplification was observed in plasma DNA.⁵⁷

Intratumoral heterogeneity of HER2 could also be demonstrated. Khoee et al.⁴⁷ showed intratumoral heterogeneity of HER2 gene amplification within cultured CTCs in 8 of 10 patients.⁵⁶ In line, Riethdorf at al. revealed intratumoral heterogeneity of HER2 amplification in CTCs of three patients. Discordance in HER2 expression within CTCs of 15 patients was detected by Gasch et al.⁵⁸

IGFR

Of ERpos breast cancers, 40%–60% express insulin-like growth factor 1 receptor (IGF1R).¹⁰⁴ In contrast, approximately 10%–20% of ERneg breast cancer express IGF1R.¹⁰⁴ The expression of IGF1R is positively correlated with the presence of ER and PR in breast cancer.¹⁰⁵ During breast cancer treatment, IGF1R expression can change in preclinical studies.^106–108 It can be downregulated by tamoxifen and upregulated by estrogen.^106–108 Drury et al.⁴⁸ showed intertumoral heterogeneity in IGF1R expression after adjuvant tamoxifen treatment. A significant reduction of IGF1R expression could be revealed in recurrent tumors after tamoxifen treatment.⁴⁸ Dhasarathy et al.⁵⁹ observed alterations in IGF1R expression of primary tumors after ET. ET decreased IGF1R expression in 11 primary tumors and increased IGF1R expression in 11 primary tumors.⁵⁹ IGF1R expression significantly decreased after treatment with AIs in primary tumor and recurrence.⁶⁰

VEGF

High VEGF levels have been linked to resistance to ET.¹⁰⁹ Intertumoral heterogeneity could be shown by Sun et al.⁵² In primary breast cancer, VEGF expression was more frequent than in metastatic lesions. Expression between primary tumor and corresponding metastasis changed in 29 of 50 patient cases.⁵²

EGFR

In preclinical studies, crosstalk between ER and epidermal growth factor receptor (EGFR) has been shown to be associated with endocrine resistance.¹¹⁰ In 1995, Cerra et al.¹¹¹ demonstrated an inverse relationship between EGFR and ER/PR status. In line, chronic fulvestrant treatment resulted in loss of ER expression and upregulation of EGFR.¹¹² EGFR amplifications and linked higher EGFR expression could be seen in triple-negative breast cancer.¹¹³

Till now, studies on inter- and intratumoral heterogeneity of EGFR receptor in breast cancer are absent. In head and neck cancer, intratumoral heterogeneity within CTCs as well as intertumoral heterogeneity between primary tumor and CTCs was revealed.¹¹⁴ In breast cancer, Nadal et al.¹¹⁵ demonstrated a significant higher proportion of patients with EGFRpos CTCs in HRpos breast cancers in comparison to HRneg primary breast cancers (33.3% vs 8.7%).

Growth factor signaling (PI3K/AKT/mTOR/FOXO)

Not only expression changes in growth factor receptors themselves, also aberrations in the growth factor signaling can lead to endocrine resistance. Growth factor signaling converges on the PI3K/AKT/mTOR pathway with PI3K as central node and the RAS/mitogen-activated protein kinase (MEK)/extracellular signal–regulated protein kinase (ERK) pathway.

In approximately 70% of breast cancers, aberrations occur in the PI3K/AKT/mTOR pathway.¹¹⁶ These include mutations or amplifications in the PI3KCA gene, in regulators of PI3K, effectors of PI3K (e.g. AKT1, AKT2, PDK1, and mTOR), and inhibitors (e.g. PTEN).¹¹⁶

PI3K

In up to 40% of breast cancers, the PI3KCA gene encoding phosphatidylinositol 3-kinase (PI3K) is mutated.^117–121

Intertumoral heterogeneity could be revealed between primary tumors, metastatic lesions, CTCs, and ctDNA. In the study of Dupont Jensen et al.,⁶¹ metastatic lesions exhibited gain of mutations in 21 cases and loss of mutations in 11 cases compared to primary tumors. In line, Gonzalez-Angulo et al.⁶² reported an 18% discordance between primary tumor and metastasis. Concerning CTCs, a disparity of 66% between primary tumor and matched CTCs was present in the study of Polzer et al.²⁶ Yu et al.⁴³ showed gain of PI3K mutations in cultured CTCs compared to corresponding primary tumor. In line, Pestrin et al.⁶³ detected PI3K mutations in CTCs of one patient which were not detectable in the matched primary tumor. Matched metastatic tumor tissue and ctDNA showed intertumoral heterogeneity. CtDNA often contained additional mutations compared to matched metastatic/primary tissue, as well as absences and vice versa.⁴⁵ PIK3CA mutations in 41 metastatic lesions/primary lesions and matched cfDNA were discordant in 5%.⁶⁴

Intratumoral heterogeneity of PI3KA mutations within primary tumors was observed in four primary breast cancers by laser capture microdissection.⁶¹ Intratumoral heterogeneity of PI3KCA mutations could be revealed within CTCs in studies of Shaw et al.,⁴² Gasch et al.,⁵⁸ Neves et al.,⁶⁵ Pestrin et al.⁶³ and Polzer et al.²⁶

AKT, mTOR, and FOXO

Mutations and alterations in expression levels of downstream effectors AKT (protein kinase B), mechanistic target of rapamycin (mTOR) and forkhead box proteins (FOXO) of PI3K signaling are reported to promote endocrine resistance.¹²²

AKT mutations have been described in 3% of breast cancer patients.^123–125 Interestingly, they were only revealed in HRpos breast cancers.^123,125 In line, activation of mTOR was reported to promote acquired resistance to ET.¹²⁶ Westin et al.¹²⁷ revealed association of mTOR activation to tamoxifen resistance. Also dysregulation of FOXO factors has emerged as key molecular feature of endocrine resistance¹²⁸ and a lack of FOXO3A expression in breast cancer patients is associated with increased recurrence rate.¹²⁹ In in vivo mouse models, FOXO inhibition promoted resistance to chemotherapy.¹²⁹ Moreover, alterations in transcriptional factors of FOXOs (e.g. E2F1) have been associated with tamoxifen resistance in ERpos breast cancer cells.¹³⁰

Until now, intra- and intertumoral heterogeneity of downstream effectors is only sparsely analyzed. Gorges et al.⁶⁶ revealed intratumoral heterogeneity of PI3K downstream effectors by multiplex transcriptome profiling of single CTCs.

PTEN

Loss of inhibitory signals from phosphatases like phosphatase and tensin homolog (PTEN) and (inositol polyphosphate 4-phosphatase type II gene (INPP4B)) affect downstream signaling and has been reported to induce resistance to ET.¹³¹ PTEN mutations affect up to 5% of breast tumors leading to dysregulation of PI3K/AKT signaling.^124,132

Intertumoral heterogeneity between primary tumor and matched metastatic lesions of PTEN could be revealed: a discordant expression of PTEN was observed in 26% of matched primary tumor and metastatic lesion.⁶² Positive PTEN primary tumors lost PTEN expression in 6% of cases, whereas 9% of PTENneg primary tumors acquired PTEN positivity at the time of recurrence.⁴⁸ Moreover, in the study of Meric-Bernstam, gain of mutation could be shown in one metastasis concerning matched primary tumor.⁶⁷

Cell-cycle alterations

Alterations in cell-cycle checkpoints can also contribute to resistance against ETs.¹³³ Cyclin-dependent kinases 4/6 (CDK4/CDK6) control the transition of the G phase to the S (synthesis) phase. Phosphorylation of retinoblastoma tumor suppressor protein (Rb) by Cyclin D1 through CDK4/CDK6 initiates the cell cycle. In general, ERpos breast cancer show a higher expression of cyclin D1 (CCND1) compared to other subtypes.^89,134 Persistent cyclin D1 expression and Rb phosphorylation are associated with ET resistance in ERpos breast cancer.¹³³

Intertumoral heterogeneity of CCND1 could be shown between primary tumor and metastasis. In the study of Meric-Bernstam et al.,⁶⁷ amplification of CCND1 was present in one metastasis but absent in its matched primary tumor. In line, this study revealed intertumoral heterogeneity of Rb and CDK4 between primary tumors and metastatic lesions. Somatic mutations of Rb (2 patients) and amplification of CDK4 (4 patients) were only seen in metastatic lesions without being detected in primary tumors.⁶⁷ Intratumoral heterogeneity of CCND1 copy numbers in cultured CTCs has also been reported.⁴⁷

Autophagy

Macroautophagy is an intracellular process by which cells recycle damaged or unnecessary organelles¹⁵ and which has been revealed to play a central role in responsiveness to ET whereas its inhibition has been linked to restoration of endocrine sensitivity.¹⁵ The unfolded protein response (UPR) is an endoplasmic reticulum stress-responsive pathway which is activated when unfolded or misfolded proteins accumulate within the endoplasmic reticulum lumen.¹³⁵ Activation of UPR pathway is associated with endocrine resistance in breast cancer.^136–138 Tamoxifen and fulvestrant can stimulate the pro-survival UPR pathway and autophagy signaling in breast cancer cells leading to endocrine resistance.¹³⁹ Glucose-regulated protein 78 (HSPA5; GRP78) is the master regulator of UPR signaling. It was shown to be increased in all breast cancer subtypes compared to normal surrounding tissue.¹⁴⁰ Overexpression of GRP78 prevented tamoxifen effectiveness, whereas knockdown of GRP78 restored ET sensitivity in in vitro models.^136,140

There are only few data regarding inter- and intratumoral heterogeneity of GRP78. In a study of Miao et al.,⁶⁸ in vivo mouse models revealed increased immunostaining of GRP78 in matched metastasis of primary breast tumors. In primary human breast cancers and matched metastasis, intertumoral heterogeneity of GRP78 expression could be also observed.⁶⁹

EMT

Epithelial breast cancer cells have the ability to transdifferentiate into motile mesenchymal cells (EMT) and vice versa (mesenchymal-to-epithelial transition (MET)). Epithelial cells reveal reduced expression of epithelial markers during EMT (e.g. loss of epithelial cytokeratins and downregulation of E-cadherin) and gain mesenchymal characteristics (e.g. upregulation of vimentin, N-cadherin).¹⁴¹ EMT can be induced by transcriptional factors, growth factor receptors, microRNA (miRNAs), epigenetic alterations and microenvironmental pressure such as hypoxia.¹⁴¹ A link between EMT and endocrine resistance could be revealed in several in vitro studies. Manipulated breast cancer cell lines mimicking cells undergoing EMT developed endocrine resistance.^142–146 Moreover, growth factor receptors (e.g. EGFR, IGF1R, and FGFR1) and transcriptional factors are involved in the induction of EMT. ERneg cells show high expression of growth factors indicating another link between EMT and endocrine resistance.¹⁴¹ In line, Dhasarathy et al. reported repression of ER by the transcriptional factor SNAIL.⁵⁹

Till now, EMT markers were predominantly studied in CTCs. CTCs expressing EMT markers seem to be an indicator for therapy-resistant cell populations.¹⁴⁷ Various studies detected EMT marker–positive CTCs in breast cancer.¹⁴⁸ Thereby, their expression within CTC pools differed between 26% and 77%.¹⁴⁸ In the study of Polioudaki et al.,⁷⁰ intratumoral heterogeneity of Vimentin was observed. In line, Bourcy also found intratumoral heterogeneity of Vimentin expression on CTCs.⁷¹ In addition to Vimentin, Kallergi et al.⁷² reported intratumoral heterogeneity of TWIST1. Intratumoral heterogeneity of N-cadherin (CDH2) expression could be shown by Armstrong et al.⁷³

Long-term navigation of endocrine-targeted therapies: a potential role for liquid biopsies?

Intra- and intertumoral heterogeneity is a major challenge in effective endocrine breast cancer treatment. Breast tumors are often composed of a dominant clone that represents the primary tumor and minor subclones. Breast tumors consisting of intra- and intertumoral heterogeneity may be more likely to contain ET-resistant subclones. If resistant subclones emerge under the current ET and outcompete the rest of the tumor cell population, real-time detection and monitoring of existing resistant subclones would be a crucial contribution to the choice of further treatment course. Although tumor or metastatic tissue is still the gold standard for molecular analyses, major barriers exist as described above. Respectively, liquid biopsies have become a noteworthy addition or alternative to tissue biopsies showing promise to overcome some of the limitations (Figure 2). Several clinical studies currently explore the role of CTCs as a prognostic and predictive marker in metastatic breast cancer patients.¹⁴⁹ Moreover, CTC count was proposed to be an indicator of ET efficacy in patients with metastatic breast cancer.¹⁷

Figure 2.

Potential role of liquid biopsies: Four proposed simplified scenarios of step-wise therapy decision-making based on availability of tissue biopsies and accurate tumor profiling. Scenario 1: performance of tissue biopsies is possible and profiling reflects representative tumor heterogeneity. Targeted therapy is effective. Scenario 2: performance of tissue biopsies is inaccurate in capturing representative tumor heterogeneity. Targeted therapy might be ineffective. Scenario 3: tissue biopsy is not possible (e.g. poor health and location). Targeted therapy is not possible and conventional therapy might be only offered. Scenario 4: missing adequate amount of tissue for profiling. Targeted therapy is not possible and conventional therapy might be only offered. In Scenarios 2, 3, and 4, profiling of “Liquid biopsies” may help as an additional clinical tool to personalize treatment.

However, additional molecular profiling of CTCs may provide important additional clinical information to CTC count (see Table 1). Molecular profiling of single CTCs holds the promise to shed light on intra- and intertumoral heterogeneity in terms of ET resistance. However, there are limitations to overcome before their implementation as clinical tools:¹⁵⁰ these are (1) the lack of standardization in defining, detecting, isolating, and molecular profiling of CTCs as well as technical restrictions when working with single cells and (2) low numbers of both CTC-pos patients and the CTC count itself in a patient which demand improved CTC isolation methods and concerted CTC storage according to harmonized standard operating procedures (SOPs). Only after these hurdles have been taken the CTCs, utility as clinical tools in detecting and sequentially monitoring ET to overcome resistance could be exploited. In analogy to CTCs, ongoing trials also investigate the potential of ctDNA extracted from plasma and serum as clinical tool. Tracking mutations through continued sampling of ctDNA may give hints for the selection for endocrine-resistant tumor subclones. In the study of Schiavon et al.,¹⁵¹ serial analysis of ctDNA revealed gain of ESR1 mutations in a patient after ET with AIs. For example, early ESR1 mutation detection may allow to discontinue a current therapy and to justify switching treatments to target ESR1 variants in order to prevent the rise of ESR1-mutated CTC subclones.

Conclusion

For more than a century, ETs have been used in the clinic to control HRpos breast cancer. Thus, many patients show primary or acquire resistance. Understanding the mechanisms that provoke endocrine resistance is essential to prolong utility of ET. In endocrine resistance, intra- and intertumoral heterogeneity of breast cancer is evident and has been demonstrated by many studies in primary tumors, metastatic lesions as well as “liquid biopsies,” that is, ctDNA and CTCs. It is imperative that we identify and monitor intra- and intertumoral heterogeneity, especially endocrine-resistant subpopulations, to restore endocrine sensitivity and prevent resistance.

To date, clinical management is based on tumor tissue biopsies and ongoing clinical trials of “liquid biopsies.” Tumor tissue biopsies are limited in many ways (see above). “Liquid biopsies” hold great promises to overcome these limitations. Being able to analyze in a repetitive and non-invasive way mutational and expression changes can help to implement patient-specific dynamic tumor landscapes. As breast tumors are composed of dynamic cellular modules generating inter- and intratumoral heterogeneity over time, ET approaches should also be dynamically adjusted based on continuous mutational and expressional profiling to prevent endocrine resistance. Therefore, continuous profiling of liquid biopsies may help as an additional clinical tool to personalize treatment methods and prevent endocrine treatment resistance in future.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Düsseldorf School of Oncology (funded by the Comprehensive Cancer Center Düsseldorf/Deutsche Krebshilfe and the Medical Faculty HHU Düsseldorf).

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