Purpose Mutations in the estrogen receptor-alpha (ER) gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Due to limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from metastatic breast cancer patients. Experimental Design We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild type ESR1 identified by next generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital polymerase chain reaction (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS and ptDNA ESR1 mutations were analyzed by ddPCR. Results In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in six of twelve patients (50%). Conclusions We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion.

Purpose Circulating Tumor cells (CTC) are prognostic in metastatic breast cancer (MBC). We tested whether the EpCAM based capture system (CellSearch®) is effective in patients with triple negative (TN) MBC, and whether CTC-apoptosis and clustering enhances the prognostic role of CTC. Experimental Design CTC enumeration and apoptosis was determined using the CXC CellSearch® kit at baseline and days 15 and 29 in blood drawn from TN MBC patients who participated in a prospective randomized phase II trial of nanoparticle albumin-bound paclitaxel (nab-PAC) with or without tigatuzumab (TIG). Association between levels of CTC and patient outcomes was assessed using logistic regression, Kaplan Meier curves, and Cox proportional hazards modeling. Results Nineteen of 52 (36.5%), 14/52 (26.9%), and 13/49 (26.5%) patients who were evaluable had elevated CTC (≥5CTC/7.5 ml WB) at baseline, days 15 and 29, respectively. Patients with elevated vs. not elevated CTC at each time point had worse progression free survival (PFS) (p=0.005, 0.0003, 0.0002, respectively). The odds of clinical benefit response for those who had elevated vs. low CTC at baseline and days 15 and 29 were 0.25 (95% CI: 0.08–0.84, p=0.024), 0.19 (95% CI: 0.05–0.17, p=0.014), and 0.06 (95% CI: 0.01–0.33, p=0.001), respectively. There was no apparent prognostic effect comparing CTC-apoptosis vs. non-apoptosis. Presence of CTC-cluster at day 15, and day 29 was associated with shorter PFS. Conclusions CTC were detected using CellSearch® assay in approximately one-third of TN MBC patients. Elevated CTC at baseline and days 15 and 29 were prognostic, and reductions in CTC levels reflected response.

Background Endocrine therapy (ET) fails to induce a response in one-half of patients with hormone receptor (HR) positive metastatic breast cancer (MBC) and almost all will eventually become refractory to ET. Circulating Tumor Cells (CTC) are associated with worse prognosis in MBC patients, but enumeration alone is insufficient to predict the absolute odds of benefit from any therapy, including ET. We developed a multi-parameter CTC-Endocrine Therapy Index (CTC-ETI), which we hypothesize may predict resistance to ET in patients with HR positive MBC. Methods The CTC-ETI combines enumeration and CTC expression of four markers: estrogen receptor (ER), B-cell lymphoma 2 (BCL-2), Human Epidermal Growth Factor Receptor 2 (HER2), and Ki67. The CellSearch® System and reagents were used to capture CTC and measure protein expression by immunofluorescent staining on CTC. Results The feasibility of determining CTC-ETI was initially established in vitro and then in a prospective single-institution pilot study in MBC patients. CTC-ETI was successfully determined in 44/50 (88%) patients. Eighteen (41%), 9 (20%), and 17 (39%) patients had low, intermediate, and high CTC-ETI scores, respectively. Inter-observer concordance of CTC-ETI determination was 94–95% (Kappa statistic 0.90–0.91). Inter- and cell-to-cell intra-patient heterogeneity of expression of each of the CTC-markers was observed. CTC biomarker expression was discordant from both primary and metastatic tissue. Conclusions CTC expression of ER, BCL-2, HER2, and Ki67 can be reproducibly measured with high analytical validity using the CellSearch® System. The clinical implications of CTC-ETI, and of the heterogeneity of CTC-biomarker expression, are being evaluated in an ongoing prospective trial.

Addressing drug resistance is a core challenge in cancer research, but the degree of heterogeneity in resistance mechanisms in cancer is unclear. In this study, we conducted next-generation sequencing (NGS) of circulating tumor cells (CTC) from patients with advanced cancer to assess mechanisms of resistance to targeted therapy and reveal opportunities for precision medicine. Comparison of the genomic landscapes of CTCs and tissue metastases is complicated by challenges in comprehensive CTC genomic profiling and paired tissue acquisition, particularly in patients who progress after targeted therapy. Thus, we assessed by NGS somatic mutations and copy number alterations (CNA) in archived CTCs isolated from patients with metastatic breast cancer who were enrolled in concurrent clinical trials that collected and analyzed CTCs and metastatic tissues. In 76 individual and pooled informative CTCs from 12 patients, we observed 85% concordance in at least one or more prioritized somatic mutations and CNA between paired CTCs and tissue metastases. Potentially actionable genomic alterations were identified in tissue but not CTCs, and vice versa. CTC profiling identified diverse intra- and interpatient molecular mechanisms of endocrine therapy resistance, including loss of heterozygosity in individual CTCs. For example, in one patient, we observed CTCs that were either wild type for ( = 5/32), harbored the known activating p.Y537S mutation ( = 26/32), or harbored a novel p.A569S ( = 1/32). p.A569S was modestly activating, consistent with its presence as a minority circulating subclone. Our results demonstrate the feasibility and potential clinical utility of comprehensive profiling of archived fixed CTCs. Tissue and CTC genomic assessment are complementary, and precise combination therapies will likely be required for effective targeting in advanced breast cancer patients. These findings demonstrate the complementary nature of genomic profiling from paired tissue metastasis and circulating tumor cells from patients with metastatic breast cancer. .

SummaryA new understanding of leaf starch degradation has emerged in the last 10 years. It has been shown that starch phosphorylation and dephosphorylation are critical components of this process. Glucan, water dikinase (GWD) (and phosphoglucan, water dikinase) adds phosphate to starch, and phosphoglucan phosphatase (SEX4) removes these phosphates. To explore the use of this metabolism to manipulate starch accumulation, Arabidopsis (Arabidopsis thaliana) plants were engineered by introducing RNAi constructs designed to reduce expression of AtGWD and AtSEX4. The timing of starch build-up was altered with ethanol-inducible and senescence-induced gene promoters. Ethanol induction of RNAi lines reduced transcript for AtGWD and AtSEX4 by 50%. The transgenic lines had seven times more starch than wild type at the end of the dark period but similar growth rates and total biomass. Elevated leaf starch content in maize leaves was engineered by making an RNAi construct against a gene in maize that appeared to be homologous to AtGWD. The RNAi construct was expressed using the constitutive ubiquitin promoter. Leaf starch content at the end of a night period in engineered maize plants was 20-fold higher than in untransformed plants with no impact on total plant biomass. We conclude that plants can be engineered to accumulate starch in the leaves with little impact on vegetative biomass.

Elevated CTC at baseline was a strong prognostic factor in this cohort. Early on-treatment changes were observed in CTC-ER and ctDNA, but not in overall CTC number. Integrating multiple biomarkers in prospective trials may improve outcome prediction and ET resistance mechanisms' identification over a single biomarker.

Fulvestrant is a dose dependent selective estrogen receptor (ER) down‐regulator (SERD) used in ER‐positive metastatic breast cancer (MBC). Nearly all patients develop resistance. We performed molecular analysis of circulating tumor cells (CTC) to gain insight into fulvestrant resistance. Preclinical studies were performed with cultured breast cancer cells spiked into human blood and analyzed on the CellSearch® system. Clinical data are limited to a subset of patients with ER‐positive MBC from a previously reported pilot trial whose disease was progressing on fulvestrant (N = 7) or aromatase inhibitors (AIs) (N = 10). CTCs were enumerated and phenotyped for ER and B‐cell lymphoma (BCL2) using the CellSearch® CXC kit. In preclinical modeling, tamoxifen and AIs resulted in stabilized ER expression, whereas fulvestrant eliminated it. Five of seven patients progressing on fulvestrant had ≥5CTC/7.5 ml WB. Two of these five, treated with 500 mg/month fulvestrant, had no detectable CTC‐expression of ER and BCL2 (an ER regulated gene). Three patients had heterogeneous CTC‐ER and BCL2 expression indicating incomplete degradation of the ER target by fulvestrant. Two of these patients received 250 mg/month whereas the third patient received 500 mg/month fulvestrant. Her cancer harbored a mutation (Y537S) in the estrogen receptor alpha gene (ESR1). All seven ER positive patients progressing on AIs had heterogeneous CTC‐ER expression. These results suggest heterogeneous mechanisms of resistance to fulvestrant, including insufficient dosage, ESR1 mutation, or conversion to dependence on non‐ER pathways. CTC enumeration, phenotyping, and genotyping might identify patients who would benefit from fulvestrant dose escalation versus switching to alternative therapies.

Circulating tumor cells (CTC) are prognostic in metastatic breast cancer (MBC). The CTC-endocrine therapy index (CTC-ETI), consisting of CTC-ER (estrogen receptor), BCL2, human epidermal growth factor receptor (HER2), and Ki67 expression, might predict resistance to endocrine therapy (ET) in patients with ER-positive MBC. One hundred twenty-one patients with ER-positive/HER2-negative MBC initiating a new ET after ≥1 lines of ET were enrolled in a prospective, multi-institutional clinical trial. CTC-ETI and clinical/imaging follow-up were performed at baseline and serial time points. Progression-free survival (PFS) and rapid progression (RP; determined at the 3-month time point) were primary endpoints. Associations with clinical outcomes used logrank and Fisher’s exact tests. At baseline, 36% (38/107) of patients had ≥5 CTC/7.5 ml whole blood (WB). Patients with ≥5 vs. <5 CTC/7.5 ml WB had significantly worse PFS (median 3.3 vs. 5.9 months, P = 0.03). Elevated CTC at 1 month was associated with even worse PFS (1.9 vs. 5.0 months from the 1-month sample, P < 0.001). Low, intermediate, and high CTC-ETI were observed in 71 (66%), 8 (8%), and 28 (26%) patients, with median PFS of 6.9, 8.5, and 2.8 months, respectively (P = 0.008). Patients with high vs. low CTC and CTC-ETI more frequently experienced RP (CTC: 66% vs. 41%; P = 0.03; CTC-ETI: 79% vs. 40%; P = 0.002). In conclusion, CTC enumeration and the CTC-ETI assay are prognostic at baseline and follow-up in patients with ER-positive/HER2-negative MBC starting new ET. CTC at first follow-up might identify a group of patients with ER-positive MBC that could forego ET, but CTC-ETI did not contribute further.