Purpose: Up to 30% of patients with breast cancer relapse after primary treatment. There are no sensitive and reliable tests to monitor these patients and detect distant metastases before overt recurrence. Here, we demonstrate the use of personalized circulating tumor DNA (ctDNA) profiling for detection of recurrence in breast cancer.Experimental Design: Forty-nine primary patients with breast cancer were recruited following surgery and adjuvant therapy. Plasma samples (n ¼ 208) were collected every 6 months for up to 4 years. Personalized assays targeting 16 variants selected from primary tumor whole-exome data were tested in serial plasma for the presence of ctDNA by ultradeep sequencing (average >100,000X).Results: Plasma ctDNA was detected ahead of clinical or radiologic relapse in 16 of the 18 relapsed patients (sensitivity of 89%); metastatic relapse was predicted with a lead time of up to 2 years (median, 8.9 months; range, 0.5-24.0 months). None of the 31 nonrelapsing patients were ctDNA-positive at any time point across 156 plasma samples (specificity of 100%). Of the two relapsed patients who were not detected in the study, the first had only a local recurrence, whereas the second patient had bone recurrence and had completed chemotherapy just 13 days prior to blood sampling.Conclusions: This study demonstrates that patientspecific ctDNA analysis can be a sensitive and specific approach for disease surveillance for patients with breast cancer. More importantly, earlier detection of up to 2 years provides a possible window for therapeutic intervention. Personalized profiling detects rising ctDNA ahead of clinical relapse. A-E, Plasma levels of ctDNA across serial plasma time points for five patients with breast cancer (one per panel). Mean VAFs are denoted by a dark blue circle, and solid lines represent the average VAF profile over time. The lead time is calculated as the time interval between clinical relapse (red triangle) and molecular relapse (blue triangle). CA 15-3 levels are graphed over time (teal circle), and the baseline levels (32 U/mL) are marked in light blue. F, Summary of percent VAF and number of targets detected at molecular and clinical relapse for all ctDNA-positive samples. Data are from 13 relapsed patients, excluding three patients with only one plasma time point. Coombes et al.

Immune checkpoint blockade (ICB) provides clinical benefit to a subset of patients with cancer. However, existing biomarkers do not reliably predict treatment response across diverse cancer types. Limited data exist to show how serial circulating tumor DNA (ctDNA) testing may perform as a predictive biomarker in patients receiving ICB. We conducted a prospective phase II clinical trial to assess ctDNA in five distinct cohorts of patients with advanced solid tumors treated with pembrolizumab (NCT02644369). We applied bespoke ctDNA assays to 316 serial plasma samples obtained at baseline and every three cycles from 94 patients. Baseline ctDNA concentration correlated with progression-free survival, overall survival, clinical response and clinical benefit. This association became stronger when considering ctDNA kinetics during treatment. All 12 patients with ctDNA clearance during treatment were alive with median 25 months follow up. This study demonstrates the potential for broad clinical utility of ctDNA-based surveillance in patients treated with ICB.

Overexpression or amplification of ACTR (also named AIB1, RAC3, p/CIP, TRAM-1, and SRC-3), a member of the p160 family of coactivators for nuclear hormone receptors, has been frequently detected in multiple types of human tumors, including breast cancer. However, its role in cancer cell proliferation and the underlying mechanism are unclear. Here, we show that overexpression of ACTR not only enhances estrogen-stimulated cell proliferation but also, more strikingly, completely negates the cell cycle arrest effect by tamoxifen and pure antiestrogens. Unexpectedly, we found that ACTR directly interacts, through its N-terminal domain, with E2F1 and is recruited to E2F target gene promoters. Elevation of ACTR in quiescent cells strongly stimulates the transcription of a subset of E2F-responsive genes that are associated with the G 1 /S transition. We also demonstrated, by adenovirus vector-mediated RNA interference, that ACTR is required for E2F1-mediated gene expression and the proliferation of estrogen receptor (ER)-negative breast cancer cells. Moreover, the ability of elevated ACTR to promote estrogen-independent cell proliferation depends on the function of E2F1 and the association between ACTR and E2F1, but not ER. Thus, our results reveal an essential role of ACTR in control of breast cancer cell proliferation and implicate the ACTR-E2F1 pathway as a novel mechanism in antiestrogen resistance.ACTR (also named AIB1, RAC3, and TRAM1; named SRC-3 and p/CIP in the mouse) is a member of the p160/SRC coactivator family (17,20,30,32). Like other p160 coactivators, ACTR/AIB1 was identified as a nuclear cofactor that associates with hormone-bound nuclear receptors and mediates the transcriptional activation function of the receptors (2,7,25,44,45). Structural and functional studies revealed that the p160s contain functional domains for histone acetyltransferase activity and interactions with ligand-bound receptors, the coregulator proteins CBP and p300, PCAF, and arginine methyltransferase CARM1. Evidence from biochemical studies suggests that the association with the above-mentioned cofactors is important for the p160s to mediate the transactivation by nuclear receptors (15,42). It is generally conceived that the p160s are recruited to hormone-responsive genes through their interaction with activated receptors and then nucleate the assembly of a coactivator complex, which in turn remodels chromatin through histone modifications and facilitates RNA polymerase II transcription. The role of ACTR/AIB1 as well as the other p160s in animal development and physiology has been explored by genetic approaches (18,35,51,56,57). Similar to the other p160 knockouts, p/CIP/SRC-3 knockout mice exhibited abnormal development and reduced function of their reproductive systems. Unlike the SRC-1 knockout mice, however, animals with the SRC-3/p/CIP gene deleted displayed significant somatic growth retardation and a reduced capacity of growth factor-stimulated cell proliferation, although the underlying molecular mechanism is unclear (51,...

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The androgen receptor (AR) is overexpressed and hyperactivated in human castration-resistant prostate cancer (CRPC). However, the determinants of AR overexpression in CRPC are poorly defined. Here we show that retinoid acid receptor-related orphan receptor γ (ROR-γ) is overexpressed and amplified in metastatic CRPC tumors, and that ROR-γ drives AR expression in the tumors. ROR-γ recruits coactivators SRC-1 and -3 to an AR-RORE to stimulate AR gene transcription. ROR-γ antagonists suppress the expression of AR and its variant AR-V7 in prostate cancer (PCa) cell lines and tumors. ROR-γ antagonists also markedly diminish genome-wide AR binding, H3K27ac abundance and expression of the AR gene network. Lastly, ROR-γ antagonists suppressed tumor growth in multiple AR-expressing but not AR-negative xenograft PCa models, and effectively sensitized CRPC tumors to enzalutamide, without overt toxicity in mice. Together, these results establish ROR-γ as a key player in CRPC by acting upstream of AR and a potential therapeutic target for advanced PCa.