Lesley-Ann Martin

Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer.

ESR1 mutations evolve during the treatment of metastatic breast cancer. An evolving problem A large number of breast cancers express the estrogen receptor, making them susceptible to hormonal treatments. Unfortunately, these tumors can develop mutations in the estrogen receptor gene (ESR1) and become resistant to hormonal therapies that were previously effective. Schiavon et al. used three independent cohorts of breast cancer patients to demonstrate that these mutations only evolved in cases where hormonal therapy was started late in the course of the disease, after development of metastasis, and not during the initial course of treatment. If these findings are confirmed in prospective clinical trials, then they will explain why starting hormonal treatment early decreases the risk of subsequent resistance to hormonal therapy. Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AIs). We developed ultra high–sensitivity multiplex digital polymerase chain reaction assays for ESR1 mutations in circulating tumor DNA (ctDNA) and investigated the clinical relevance and origin of ESR1 mutations in 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies and was accurately assessed in samples shipped at room temperature in preservative tubes. ESR1 mutations were found exclusively in estrogen receptor–positive breast cancer patients previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy [hazard ratio, 3.1; 95% confidence interval (CI), 1.9 to 23.1; P = 0.0041]. ESR1 mutation prevalence differed markedly between patients who were first exposed to AI during the adjuvant and metastatic settings [5.8% (3 of 52) versus 36.4% (16 of 44), respectively; P = 0.0002]. In an independent cohort, ESR1 mutations were identified in 0% (0 of 32; 95% CI, 0 to 10.9) tumor biopsies taken after progression on adjuvant AI. In a patient with serial sampling, ESR1 mutation was selected during metastatic AI therapy to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI but are commonly selected by therapy for metastatic disease, providing evidence that mechanisms of resistance to targeted therapy may be substantially different between the treatment of micrometastatic and overt metastatic cancer.

Genetic Prodrug Activation Therapy for Breast Cancer: A Phase I Clinical Trial of erbB-2–Directed Suicide Gene Expression

PURPOSE This trial was designed to test the safety and efficacy of a tumor-specific genetic prodrug activation therapy targeted by use of the human erbB-2 gene promoter. The erbB-2 oncogene is overexpressed in approximately 20% of cases of breast cancer and is associated with poor prognosis. PATIENTS AND METHODS Twelve breast cancer patients received transcriptionally targeted gene therapy in a phase I clinical trial using direct intratumoral injection of plasmid construct combined with systemic administration of prodrug. The genetic prodrug activation therapy is specifically targeted to erbB-2-overexpressing breast cancer cells by use of a therapeutic cassette that contains the Escherichia coli cytosine deaminase gene driven by the tumor-specific erbB-2 promoter, thus allowing activation of fluorocytosine to the active cytotoxic fluorouracil only within tumor cells that express the oncogene. RESULTS The approach was shown to be safe and to result in targeted gene expression in up to 90% of cases. Using a number of different assays, we demonstrated that significant levels of expression of the suicide gene were specifically restricted to erbB-2-positive tumor cells, confirming the selectivity of the approach. CONCLUSION The results of this study, the first targeted gene therapy for breast cancer and the first to use the cytosine deaminase system in human subjects, are encouraging for the development of genetic prodrug activation therapies that exploit the transcriptional profile of cancer cells.

Molecular changes associated with the acquisition of oestrogen hypersensitivity in MCF-7 breast cancer cells on long-term oestrogen deprivation.

The growth dependence of many breast cancers on oestrogen has been exploited therapeutically by oestrogen deprivation, but almost all patients eventually develop resistance largely by unknown mechanisms. Wild-type (WT) MCF-7 cells were cultured in oestrogen-deficient medium for 90 weeks in order to establish a long-term oestrogen-deprived MCF-7 (LTED) which eventually became independent of exogenous oestrogen for growth. After 15 weeks of quiescence (LTED-Q), basal growth rate increased in parallel with increasing oestrogen sensitivity. While 10(-9)M oestradiol (E2) maximally stimulated WT growth, the hypersensitive LTED (LTED-H) were maximally growth stimulated by 10(-13)M E2. By week 50, hypersensitivity was apparently lost and the cells became oestrogen independent (LTED-I), although the pure antioestrogen ICI182780 still inhibited cell growth and reversed the inhibitory effect of 10(-9)M E2 at 10(-12) to 10(-7)M. Tamoxifen (10(-7) to 10(-6)M) had a partial agonist effect on WT, but had no stimulatory effect on LTED. Whilst LTED cells have a low progesterone receptor (PgR) expression in all phases, oestrogen receptor (ER) a expression was, on average, elevated five- and seven-fold in LTED-H and LTED-I, respectively, and serine118 was phosphorylated. ERbeta expression was up-regulated and the levels of insulin receptor substrate 1 (IRS-1) remained low throughout all phases. The levels of RIP140mRNA appeared to decrease to approximately 50% of the WT message in LTED-Q and remained constant into the hypersensitive phase. No significant changes were observed in the expression of SUG-1, TIF-1 and SMRT in LTED. The overall changes in nuclear receptor interacting proteins do not appear to be involved in the hypersensitivity. Thus, the resistance of these human breast cancer cells to oestrogen-deprivation appears to be due to acquired hypersensitivity which may be explained in part by increased levels of and phosphorylated ERalpha.

Mechanisms of disease: angiogenesis and the management of breast cancer.

Demonstration of the clinically significant activity of bevacizumab in breast cancer has attracted a great deal of interest. Numerous other antiangiogenic treatments are in clinical development and some established therapies including tamoxifen and trastuzumab might function, in part, by suppressing angiogenesis. In this Review, we discuss the potential of various components of the angiogenic pathway as prognostic and predictive factors in breast cancer. In addition, we describe existing clinical trials of antiangiogenic agents and the challenges facing the clinical development and optimum use of these agents for the treatment of breast cancer.

Biomarkers for the clinical management of breast cancer: International perspective

The higher incidence of breast cancer in developed countries has been tempered by reductions in mortality, largely attributable to mammographic screening programmes and advances in adjuvant therapy. Optimal systemic management requires consideration of clinical, pathological and biological parameters. Oestrogen receptor alpha (ERα), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (HER2) are established biomarkers evaluated at diagnosis, which identify cardinal subtypes of breast cancer. Their prognostic and predictive utility effectively guides systemic treatment with endocrine, anti‐HER2 and chemotherapy. Hence, accurate and reliable determination remains of paramount importance. However, the goals of personalized medicine and targeted therapies demand further information regarding residual risk and potential benefit of additional treatments in specific circumstances. The need for biomarkers which are fit for purpose, and the demands placed upon them, is therefore expected to increase. Technological advances, in particular high‐throughput global gene expression profiling, have generated multi‐gene signatures providing further prognostic and predictive information. The rational integration of routinely evaluated clinico‐pathological parameters with key indicators of biological activity, such as proliferation markers, also provides a ready opportunity to improve the information available to guide systemic therapy decisions. The additional value of such information and its proper place in patient management is currently under evaluation in prospective clinical trials. Expanding the utility of biomarkers to lower resource settings requires an emphasis on cost effectiveness, quality assurance and possible international variations in tumor biology; the potential for improved clinical outcomes should be justified against logistical and economic considerations.

Targeting the receptor tyrosine kinase RET sensitizes breast cancer cells to tamoxifen treatment and reveals a role for RET in endocrine resistance

Endocrine therapy is the main therapeutic option for patients with estrogen receptor (ERα)-positive breast cancer. Resistance to this treatment is often associated with estrogen-independent activation of ERα. In this study, we show that in ERα-positive breast cancer cells, activation of the receptor tyrosine kinase RET (REarranged during Transfection) by its ligand GDNF results in increased ERα phosphorylation on Ser118 and Ser167 and estrogen-independent activation of ERα transcriptional activity. Further, we identify mTOR as a key component in this downstream signaling pathway. In tamoxifen response experiments, RET downregulation resulted in 6.2-fold increase in sensitivity of MCF7 cells to antiproliferative effects of tamoxifen, whereas GDNF stimulation had a protective effect against the drug. In tamoxifen-resistant (TAMR-1) MCF7 cells, targeting RET restored tamoxifen sensitivity. Finally, examination of two independent tissue microarrays of primary human breast cancers revealed that expression of RET protein was significantly associated with ERα-positive tumors and that in primary tumors from patients who subsequently developed invasive recurrence after adjuvant tamoxifen treatment, there was a twofold increase in the number of RET-positive tumors. Together these findings identify RET as a potentially important therapeutic target in ERα-positive breast cancers and in particular in tamoxifen-resistant tumors.

Lapatinib Restores Hormone Sensitivity with Differential Effects on Estrogen Receptor Signaling in Cell Models of Human Epidermal Growth Factor Receptor 2–Negative Breast Cancer with Acquired Endocrine Resistance

Purpose: Acquired endocrine resistance in estrogen receptor (ER)α+/human epidermal growth factor receptor 2–negative (HER2−) breast cancer has been associated with modest adaptive increases in HER2, although exactly how aberrant HER2 signaling affects the ERα pathway is poorly understood. We investigated (a) whether the epidermal growth factor receptor/HER2 inhibitor lapatinib could restore endocrine responsiveness in cell models of acquired endocrine resistance with modest increases in HER2, and (b) the nature of ERα-HER2 cross-talk in this process. Methods: Combination growth studies, ERα transcription, immunoblot, and gene expression assays were conducted in two models of acquired resistance to (a) estrogen deprivation (long-term estrogen-deprived cells) and (b) tamoxifen (long-term tamoxifen-treated cells), and in hormone sensitive controls. Changes in ERα, PgR, and HER2 were assessed in samples from patients treated with tamoxifen. Results: Both cell models of acquired endocrine resistance showed modest adaptive upregulation in HER2, and lapatinib restored endocrine sensitivity in both. The effect of lapatinib on ERα signaling varied markedly depending on the nature of the HER2/ERα cross-talk. In long-term estrogen-deprived cells characterized by enhanced ERα function, lapatinib suppressed ERα genomic activity (as measured by pERSer118, ERα transcriptional activity, and PGR gene expression). In contrast, in long-term tamoxifen-treated cells with reduced ERα activation, lapatinib reactivated ERα genomic function. Twenty percent of tamoxifen-resistant patients relapsed with modest increases in HER2 and either suppressed or enhanced ERα/PgR expression. Conclusions: Aberrant GFR signaling can augment or suppress ERα function. Regardless, interrupting the HER2/ERα cross-talk with lapatinib can restore endocrine sensitivity and should be investigated as a therapeutic strategy in combination with endocrine therapy in ERα+/HER2− patients with acquired endocrine resistance. Clin Cancer Res; 16(5); 1486–97

Early Adaptation and Acquired Resistance to CDK4/6 Inhibition in Estrogen Receptor-Positive Breast Cancer

Small-molecule inhibitors of the CDK4/6 cell-cycle kinases have shown clinical efficacy in estrogen receptor (ER)-positive metastatic breast cancer, although their cytostatic effects are limited by primary and acquired resistance. Here we report that ER-positive breast cancer cells can adapt quickly to CDK4/6 inhibition and evade cytostasis, in part, via noncanonical cyclin D1-CDK2-mediated S-phase entry. This adaptation was prevented by cotreatment with hormone therapies or PI3K inhibitors, which reduced the levels of cyclin D1 (CCND1) and other G1-S cyclins, abolished pRb phosphorylation, and inhibited activation of S-phase transcriptional programs. Combined targeting of both CDK4/6 and PI3K triggered cancer cell apoptosis in vitro and in patient-derived tumor xenograft (PDX) models, resulting in tumor regression and improved disease control. Furthermore, a triple combination of endocrine therapy, CDK4/6, and PI3K inhibition was more effective than paired combinations, provoking rapid tumor regressions in a PDX model. Mechanistic investigations showed that acquired resistance to CDK4/6 inhibition resulted from bypass of cyclin D1-CDK4/6 dependency through selection of CCNE1 amplification or RB1 loss. Notably, although PI3K inhibitors could prevent resistance to CDK4/6 inhibitors, they failed to resensitize cells once resistance had been acquired. However, we found that cells acquiring resistance to CDK4/6 inhibitors due to CCNE1 amplification could be resensitized by targeting CDK2. Overall, our results illustrate convergent mechanisms of early adaptation and acquired resistance to CDK4/6 inhibitors that enable alternate means of S-phase entry, highlighting strategies to prevent the acquisition of therapeutic resistance to these agents. Cancer Res; 76(8); 2301-13. ©2016 AACR.

ERBB2 influences the subcellular localization of the estrogen receptor in tamoxifen-resistant MCF-7 cells leading to the activation of AKT and RPS6KA2.

Acquired resistance to endocrine therapies remains a major clinical obstacle in hormone-sensitive breast tumors. We used an MCF-7 breast tumor cell line (Tam(R)-1) resistant to tamoxifen to investigate this mechanism. We demonstrate that Tam(R)-1 express elevated levels of phosphorylated AKT and MAPK3/1-activated RPS6KA2 compared with the parental MCF-7 cell line (MCF-7). There was no change in the level of total ESR between the two cell lines; however, the Tam(R)-1 cells had increased phosphorylation of ESR1 ser(167). SiRNA blockade of AKT or MAPK3/1 had little effect on ESR1 ser(167) phosphorylation, but a combination of the two siRNAs abrogated this. Co-localization studies revealed an association between ERBB2 and ESR1 in the Tam(R)-1 but not MCF-7 cells. ESR1 was redistributed to extranuclear sites in Tam(R)-1 and was less transcriptionally competent compared with MCF-7 suggesting that nuclear ESR1 activity was suppressed in Tam(R)-1. Tamoxifen resistance in the Tam(R)-1 cells could be partially overcome by the ERBB2 inhibitor AG825 in combination with tamoxifen, and this was associated with re-localization of ESR1 to the nucleus. These data demonstrate that tamoxifen-resistant cells have the ability to switch between ERBB2 or ESR1 pathways promoting cell growth and that pharmacological inhibition of ERBB2 may be a therapeutic strategy for overcoming tamoxifen resistance.

Mechanisms of resistance to aromatase inhibitors

Aromatase inhibitors are rapidly becoming the first choice for hormonal treatment of steroid receptor positive breast cancer in postmenopausal women. An understanding of the resistance mechanisms to these agents is, therefore, important for the appropriate delivery of treatment to responsive patients and the rational development of new agents targeted at the resistance pathways. De novo resistance appears to be a quantitative rather than qualitative phenomenon with virtually all oestrogen receptor positive tumours showing an anti-proliferative response to the aromatase inhibitor anastrozole. While the expression of type 1 growth factor receptors reduces response to tamoxifen this appears to have little detrimental effect on response to aromatase inhibitors. Studies of acquired resistance in vitro have indicated that acquisition of hypersensitivity to oestrogenic stimulation is a key mechanism that is dependent on enhanced cross-talk of growth factor and oestrogen signaling pathways. Collection of resistant biopsy tissues from patients is important to determine if this mechanism is clinically relevant.