Heterogeneity of estrogen receptor expression in circulating tumor cells from metastatic breast cancer patients

Abstract

Background: Endocrine treatment is the most preferable systemic treatment in metastatic breast cancer patients that have had an estrogen receptor (ER) positive primary tumor or metastatic lesions, however, approximately 20% of these patients do not benefit from the therapy and demonstrate further metastatic progress. One reason for failure of endocrine therapy might be the heterogeneity of ER expression in tumor cells spreading from the primary tumor to distant sites which is reflected in detectable circulating tumor cells (CTCs).

Methods: A sensitive and specific staining protocol for ER, keratin 8/18/19, CD45 was established. Peripheral blood from 35 metastatic breast cancer patients with ER-positive primary tumors was tested for the presence of CTCs. Keratin 8/18/19 and DAPI positive but CD45 negative cells were classified as CTCs and evaluated for ER staining. Subsequently, eight individual CTCs from four index patients (2 CTCs per patient) were isolated and underwent whole genome amplification and ESR1 gene mutation analysis.

Results: CTCs were detected in blood of 16 from 35 analyzed patients (46%), with a median of 3 CTCs/7.5 ml. In total, ER-negative CTCs were detected in 11/16 (69%) of the CTC positive cases, including blood samples with only ER-negative CTCs (19%) and samples with both ER-positive and ER-negative CTCs (50%). No correlation was found between the intensity and/or percentage of ER staining in the primary tumor with the number and ER status of CTCs of the same patient. ESR1 gene mutations were not found.

Conclusion: CTCs frequently lack ER expression in metastatic breast cancer patients with ER-positive primary tumors and show a considerable intra-patient heterogeneity, which may reflect a mechanism to escape endocrine therapy. Provided single cell analysis did not support a role of ESR1 mutations in this process.

Figure 1. Triple immunostaining of a metastatic breast cancer patient blood sample.

From left to right: estrogen receptor (ER) stained with AlexaFluor 488 (green), keratins 8/18/19 (K) stained with Cy3 (red), DAPI (fluorescent blue) for counter staining, CD45, stained with NBT/BCIP (visible dark blue), and all channels merged. Magnification x100. Row A. Images of ER-positive CTC. A cell with phenotype ER+ (green)/ K+ (red)/CD45- (dark blue)/DAPI+ (fluorescent blue) is considered to be ER-positive CTC. CTC is surrounded with leukocytes (phenotype ER-/K-/CD45+/DAPI+). Row B. Images of ER-negative CTC. A single CTC demonstrating no specific nuclear ER staining. The phenotype is ER-/K+/CD45-/DAPI+. Leukocytes present ER-/K-/CD45+/DAPI+ phenotype.

Figure 2. Kaplan–Meier estimate of survival function.

Kaplan–Meier estimate of survival function of metastatic breast cancer patients separated on CTC-positive (red line) and CTC-negative (blue line) groups. The survival period in month of the corresponding patient. Censored patients are indicated by vertical bars (|). Statistical significance determined by log-rank test. Shorter survival correlates with presence of CTCs in blood (P: 0.0332, HR: 7.38, (CI = 0.84-64.09)).

Figure 3. Occurrence of ER-positive and ER-negative CTCs in the peripheral blood of patients with breast carcinomas classified as ER-positive.

Circulating tumor cells disseminating from an ER-positive breast tumor can be ER-positive or ER-negative. ER-positive CTCs can have normal functional ER machinery and be sensitive to endocrine therapy (cell A) or have dysfunctional ER machinery and therefore be resistant to endocrine therapy (cell B). ER-negative CTCs might disseminate from ER-negative subclones in tumors classified as ER-positive (diagnostic cut-off value: 1% of ER-stained tumor cells) (cell C) or disseminate from ER-positive subclones that lost ER expression during the metastatic cascade or as a result of systemic therapy (cell D).