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

Abstract

BackgroundEndocrine 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).MethodsA 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.ResultsCTCs 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.ConclusionCTCs 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.

Acoustic separation of circulating tumor cells

“…In the first patient immunohistochemical staining of a biopsy of a metastatic site (bone) showed persistent ER positivity and progesterone receptor (PR) positivity at 5%; the second patient had biopsy of her metastatic tumor in the pelvis and this was ER-positive at 100% and PRpositive at 1%. Loss of ER positivity in the CTCs from patients with initial diagnosis of ER positive breast cancer has been previously reported and could be a reflection of tumor heterogeneity (16). The third patient had a low (1) CTC count, which is consistent with the fact that the blood was drawn within 2 mo after initiating a new line of endocrine therapy to which she responded; this was ascertained by clinical examination and imaging (CT scans).…”

Multiplexed single cell protein expression analysis in solid tumours using a miniaturised microfluidic assay

“…For example, a HER2 assay could examine expression level heterogeneity in HER2 positive breast or gastric cancer and could be assessed for correlation with treatment outcomes for trastuzumab, an anti-HER2 antibody drug, which is routinely applied to these patients. Furthermore, receptor expression heterogeneity in circulating tumour cells (CTCs) has been identified as a possible mechanism of acquired resistance to treatments targeting estrogen receptor and epidermal growth factor receptor (EGFR) in breast and colorectal cancer respectively [45,46]. Characterising this heterogeneity with high resolution is important for the identification of clear subpopulations, and this is something MAC chips can address in CTCs as well as the tumour itself.…”

Clinical Applications of Circulating Tumor Cells and Circulating Tumor DNA as Liquid Biopsy

“…However, ER-positive breast tumors can harbor ER-negative CTCs, which might be able to escape endocrine therapy ( 55 Another target, the HER2 oncogene, which is amplifi ed and overexpressed in approximately 20% of primary carcinomas, has become a key target in breast cancer. There is increasing evidence that overt distant metastases and CTCs have discrepant HER2 statuses compared to the primary tumor in up to 30% of cases ( 57 ).…”