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.
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.
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