Heterogeneity of Estrogen Receptor Expression in Circulating Tumor Cells from Metastatic Breast Cancer Patients

<div><p>Background</p><p>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).</p><p>Methods</p><p>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 <i>ESR1</i> gene mutation analysis.</p><p>Results</p><p>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. <i>ESR1</i> gene mutations were not found.</p><p>Conclusion</p><p>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 <i>ESR1</i> mutations in this process.</p></div

Kaplan–Meier estimate of survival function.

<p>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 (<i>P:</i> 0.0332, HR: 7.38, (CI = 0.84-64.09)).</p

The established triple staining protocol for detection and characterization of ER expression on CTC.

<p>ER – estrogen receptor; CTC – circulating tumor cell; NBT/BCIP – nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate; PBS – phosphate buffered saline; TBS – tris buffered saline.</p

Overview of studies on ER status of CTC in metastatic breast cancer patients.

<p>CTC – circulating tumor cell; ER – estrogen receptor; IF – immunofluorescence; RT-PCR – real-time PCR.</p>*<p>RT-PCR approach does not allow to assess intrapatient heterogeneity of ER-status of CTCs.</p

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

<p>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).</p

CTC status in respect to clinical status of the patients.

<p>CTC – circulating tumor cell.</p

Number of detected CTCs and corresponding ER status.

*<p>ER positive group includes CTCs with weak, moderate, and strong uniform ER staining. For more detailed information see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075038#pone.0075038.s001" target="_blank">Table S1</a>.</p><p>ER – estrogen receptor; CTC – circulating tumor cell.</p

Kaplan-Meier curves for overall survival according to the CTC status using the AdnaTest^® (A), CellSearch^® (B), or both assay in combination (C).

<p>Kaplan-Meier curves for overall survival according to the CTC status using the AdnaTest^® (A), CellSearch^® (B), or both assay in combination (C).</p

Patient characteristics.

<p>Patient characteristics.</p

Kaplan-Meier curves for progression-free survival according to the CTC status using the AdnaTest^® (A), CellSearch^® (B), or both assay in combination (C).

<p>Kaplan-Meier curves for progression-free survival according to the CTC status using the AdnaTest^® (A), CellSearch^® (B), or both assay in combination (C).</p