B E T W E E N B E D S I D E A N D B E N C H

Ovarian hormones such as estrogens play a key part in normal mammary gland development and in breast tumorigenesis. Estrogens medi-ate their action on mammary epithelial cells via estrogen receptor (ER), a ligand-dependent nuclear steroid hormone receptor encoded by the ESR1 gene. Because the majority (~70%) of human breast tumors are ER positive and depend on estrogen for growth, the use of selective ER modulators, such as tamoxifen, in ER-expressing tumors was one of the first examples for successful targeted therapy based on the molecular classification of tumors1. Better understanding of ER function led to the development of multiple other targeted agents with different mechanisms of action, allow-ing for the more effective inhibition of the ER signaling pathway and fewer side effects. Unfortunately, about a third of ER-positive breast cancers are inherently resistant to endo-crine therapy, although they may still respond to other drugs with different mechanisms of action, and 30–40% of the initial responders will also eventually progress to resistant disease. Because the options for these patients are lim-ited, understanding what induces endocrine resistance in these tumors has been one of the longest standing and most intense areas of breast cancer research.

Multiple molecular alterations have been shown to mediate endocrine resistance through different mechanisms including activation of kinase signaling pathways, altered expression

of cell cycle regulators, overexpression of ER coactivators, intratumor heterogeneity of ER, and activation of epithelial to mesenchymal transition (EMT)2. Contrary to findings with resistance to other types of targeted therapies, genetic alterations in the target itself, ESR1, have not been found. And although rare ESR1 mutations have been found in human breast and endometrial carcinomas, most of these have unclear functional relevance3,4 or are thought to be too infrequent to explain the major fraction of endocrine resistance5. Even the Cancer Genome Atlas Research Network (TCGA) failed to identify ESR1 mutations in recent comprehensive breast-cancer-genome sequencing human studies6. Nonetheless, this all changed in the past few months with a flurry of papers reporting common mutations in ESR1 ligand-binding domain in endocrine-resistant metastatic human breast carcinomas, implying a direct role for these mutations in therapeutic resistance7–11.

The first hint that ESR1 mutations may under-lie endocrine resistance came from Li et al.7 who established and characterized patient-derived xenograft models of breast cancer to be used for preclinical studies. Whole-genome sequenc-ing of patient-derived xenografts derived from treatment-resistant metastatic tumors revealed frequent genetic alterations—amplification, translocation, and point mutation—in ESR1, which correlated with the estrogen-independent growth of these xenografts. Exogenous expres-sion of these mutants in ER-positive breast cancer cell lines also conferred estrogen inde-pendence and resistance to endocrine therapy and was associated with constitutive activa-tion of classic direct ER target genes, such as PGR (encoding the progesterone receptor) and

TFF1 (encoding trefoil factor 1). Notably, deep sequencing of the original patient sample also confirmed the presence of these mutations.

Similarly, four independent studies analyz-ing human samples of metastatic breast cancer from different cohorts by whole-exome or tar-geted cancer gene sequencing reported essen-tially the same findings: ESR1 ligand-binding domain mutations occur in a substantial frac-tion (10–30%) of endocrine-resistant metastatic disease8–11. Two of the cohorts were small (11 and 13 patients, respectively), and even the larger ones included only about 80 cases, mak-ing it difficult to conclude statistically significant associations with clinical-pathological and other molecular features. However, all of the mutant cases had received multiple lines of endocrine therapies such as tamoxifen and aromatase inhibitors, and almost all continued expressing ER despite disease progression.

All four studies also showed that the ESR1 mutations lead to ligand-independent activa-tion of ER targets and decrease sensitivity to endocrine therapies in cell culture models. Unfortunately, matched primary pretreat-ment samples were only available for a few cases, and in none of these was the mutation detected at the sequencing depth analyzed (20×–500× coverage), suggesting that ESR1 mutations were acquired during disease pro-gression, and, even if present before treatment, the mutant cells were a very small fraction of the original tumor (Fig. 1a). These results can also explain why such ESR1 mutations have not been described by any of the other large-scale sequencing studies focusing on treatment-naive primary breast tumors.

As with many new discoveries, the results raise more questions than answers. One of

Kornelia Polyak is at the Department of Medical

Oncology, Dana-Farber Cancer Institute, and

Department of Medicine, Harvard Medical School,

Boston, Massachusetts, USA.

e-mail: kornelia_polyak@dfci.harvard.edu

■ BENCH TO BEDSIDE

A case for Darwinian tumor evolutionKornelia Polyak

It is clear that innovations in genomic technology and bioinformatics analysis have afforded an unprecedented view of the somatic cancer genome and have illuminated the breadth of genetic diversity within tumors. The next challenge is to determine how clonal heterogeneity informs the biology of differ-ent tumors and to assess whether delineating clonal architecture in primary tumors can serve as a biomarker for clinical outcome or for therapeutic response. Only through such

studies can we move beyond our current view of cancer heterogeneity as a ‘black box’ and begin to use our understanding of this impor-tant concept to improve outcomes for patients with cancer.

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

1. Vogelstein, B. et al. Science 339, 1546–1558 (2013).2. Ciriello, G. et al. Nat. Genet. 45, 1127–1133 (2013).3. Fearon, E.R. & Vogelstein, B. Cell 61, 759–767

(1990).

4. Patel, J.P. et al. N. Engl. J. Med. 366, 1079–1089 (2012).

5. Lawrence, M.S. et al. Nature 499, 214–218 (2013).6. Ding, L. et al. Nature 481, 506–510 (2012).7. Walter, M.J. et al. N. Engl. J. Med. 366, 1090–1098

(2012).8. Gerlinger, M. et al. N. Engl. J. Med. 366, 883–892

(2012).9. Magrangeas, F. et al. Leukemia 27, 473–481 (2013).10. Landau, D.A. et al. Cell 152, 714–726 (2013).11. Clappier, E. et al. J. Exp. Med. 208, 653–661 (2011).12. Notta, F. et al. Nature 469, 362–367 (2011).13. Papaemmanuil, E. et al. Blood 122, 3616–3627

(2013).14. Lohr, J.G. et al. Cancer Cell 25, 91–101 (2014).

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B E T W E E N B E D S I D E A N D B E N C H

the most obvious ones is whether the ligand-domain ESR1 mutants are simply activating the same ER target genes, as wild-type ER does, in a ligand-independent manner or whether they have some additional gain-of-function phe-notype. The gene expression profiles of MCF7 cells transfected with the various mutants8 and the increased migration and invasion of these cells11 indicate that mutant ESR1 also renders cells more mesenchymal, potentially by turn-ing on downstream targets of some growth factor signaling (Fig. 1b). Thus, in a way, mutant ESR1-associated endocrine resistance resembles that caused by activation of EMT and receptor tyrosine kinase pathways such as increased AKT or mitogen-activated pro-tein kinase signaling, which can occur within the same tumor. The observation that tumors with ESR1 mutation had a lower frequency of PIK3CA mutation7,11 also supports the hypoth-esis that mutant ESR1 activates both ER and growth factor signaling targets, although the number of samples analyzed is too small to make any definitive conclusions regarding interactions between ESR1 and other muta-tions found in the tumors.

Another question is whether the ESR1-mutant cells can be found in all metastatic lesions within one individual with breast cancer, which would imply shared subclonal origin, or whether they are selected in specific microenvi-ronments. One of the studies analyzed two dif-ferent metastases from the same patient—liver and lung—but only detected ESR1 mutations in the liver metastasis11. However, given the diver-sity of the metastatic lesions where mutations have been found (i.e., skin, lung, liver, bone and lymph nodes), the presence of ESR1-mutant cells is probably widespread and not limited to particular organ sites.

Lastly, what is the clinical relevance of ESR1 mutations with regards to prognosis and response to treatment, and how could we use them for the development of better selective ER modulators and more effective therapies? Toy et al.8 analyzed patient tumors from the BOLERO-2 clinical trial12, which compared the standard treatment exemestane (an aroma-tase inhibitor) with a combination of exemes-tane and the mammalian target of rapamycin inhibitor everolimus. They found that only 3% of the primary tumors before treatment had

ESR1 mutation, compared to 11% of meta-static tumors that progressed during treatment, strongly suggesting selection for cells with mutant ESR1 during treatment. Furthermore, the progression-free survival for ESR1-mutant cases was not different between the two arms of the study, which is not surprising as the number of patients with ESR1-mutant tumors was very low and, owing to the trial design, all patients had already progressed on a nonsteroidal aro-matase inhibitor treatment12. Thus, analysis of larger cohorts is required to determine whether combining growth factor signaling pathway inhibitors with endocrine therapy would be more effective against tumors with ESR1 muta-tion.

What seems to be clear is that ESR1-mutant cells can still be inhibited by selective ER mod-ulators and selective ER downregulators, such as fulvestrant; however, these cells required higher concentrations than tumor cells lack-ing the ESR1 mutation7–9,11. This would imply that the design of more potent selective ER modulators and their use at higher doses might be required for treatment of ESR1-mutant tumors, as suggested by the results of

Figure 1 The role of ESR1 mutations in breast cancer endocrine resistance. (a) Despite multiple lines of endocrine therapies, selection for cancer cells with ESR1 ligand-binding domain (LBD) mutations during disease progression occurs, leading to resistant disseminated breast cancers. (b) Wild-type ER is activated by estradiol (E2), leading to its association with various coactivators (CoA) and the activation of ER target genes required for cell proliferation and survival. In contrast, LBD-mutant ER does not require E2 for its activity, it may associate with coactivators that are different than those used by wild-type protein, and it may activate both ER targets and downstream targets of growth factor signaling pathways. RTK, receptor tyrosine kinases; TF, transcription factors.

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therapies have led to better inhibition, but also stronger selection for cells with ESR1 muta-tions that are less sensitive to estrogen-blocking agents. Related to this is the observation that alternating high-dose estrogen with estrogen withdrawal may in some cases decrease disease progression and prolong survival14, poten-tially because of alteration of the population dynamics and subclonal interactions between estrogen-dependent and estrogen-independent tumor subclones.

One of the most exciting outcomes of com-prehensive cancer-genome-sequencing stud-ies, such as the ones that led to the discovery of ESR1 mutant tumor cells, is that we finally have the tools to follow clonal and subclonal evolution of tumors and see the complexity of cancers as a whole. Tumor progression is driven by Darwinian-like somatic evolution15; under-standing the subclonal architecture of tumors should finally allow researchers to incorporate the evolutionary perspective into the design of more accurate preclinical models that reflect tumor heterogeneity. The use of such models and the analysis of clinical samples from a more evolutionary view will hopefully also lead to

the CONFIRM trial that showed improved overall survival with higher doses of fulves-trant compared to the low-dose group in post-menopausal women with locally advanced or metastatic ER-positive breast cancers that were refractory to endocrine therapy13. However, because these ESR1 mutations are not detected in the primary tumors before treatment, patient stratification would require the testing of emerging metastatic lesions. Nevertheless, preventing disease progression would defi-nitely be preferred. It would also be interest-ing to see whether sequencing of circulating tumor cells (CTCs), bone marrow biopsies or plasma DNA might be used to detect ESR1 mutations before metastatic disease becomes clinically obvious.

Would increasing selection pressure with more potent drugs actually improve treatment or only accelerate the expansion of ESR1-mutant subclones at earlier stages of disease? One of the reasons ESR1-mutant clones have not been detected in the past could poten-tially be owing to the relative inability of early endocrine treatments to effectively block the ER signaling pathway. Improved endocrine

B E T W E E N B E D S I D E A N D B E N C H

changes in the design of cancer therapies, which will result in more effective eradiation or at least better control of tumors.

COMPETING FINANCIAL INTERESTS The author declares no competing financial interests.

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(2013).10. Jeselsohn, R. et al. Clin. Cancer Res. doi:10.1158/1078-

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