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

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

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

Patients' characteristics and correlation of serum RNA and relative levels of circulating miRs with clinical and histopathological parameters of the validation cohort.

α<p>p = 0.048,</p>β<p>p = 0.014,</p>ε<p>p = 0.030,</p>δ<p>p = 0.031;</p>£<p>M0, patients with localized NSCLC;</p>$<p>M1, patients with metastatic NSCLC;</p>#<p>M0 patients;</p><p>p values as determined by Mann Whitney-U test.</p

Example of a loss of heterozygosity (LOH) detected in blood serum from a patient with breast cancer

<p><b>Copyright information:</b></p><p>Taken from "A critical evaluation of loss of heterozygosity detected in tumor tissues, blood serum and bone marrow plasma from patients with breast cancer"</p><p>http://breast-cancer-research.com/content/9/5/R66</p><p>Breast cancer research : BCR 2007;9(5):R66-R66.</p><p>Published online 3 Oct 2007</p><p>PMCID:PMC2242661.</p><p></p> The fluorescence-labeled PCR products of leukocytes and serum DNA were separated by capillary gel electrophoresis on a Genetic Analyzer and evaluated with the Gene Scan Analysis program. The abscissa indicates the length of the PCR product, while the ordinate gives information on the fluorescence intensity represented as peaks. The upper and lower diagram show the leukocyte DNA (reference) and serum DNA amplified with the primer binding at the D17S855. The PCR product of the serum DNA shows a LOH

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

Evaluation of the diagnostic relevance of levels of total RNA and miRs in serum of healthy individuals, patients with benign lung disease and NSCLC patients.

<p>The box plots show the different, relative amounts of total RNA (A), miR-361-3p (B) and miR-625* (C) which circulate in blood of healthy individuals (n = 30), patients with benign lung disease (n = 20) and NSCLC patients (n = 97). The relative transcript levels of miRs were determined by the low cycle threshold (Ct) values. As determined by Mann and Whitney-U test, the significant p values of the statistical evaluations of serum RNA and miR levels are indicated. The ROC analysis shows the profile of sensitivity and specificity of miR-361-3p and miR-625* concentrations to discriminate NSCLC patients from patients with benign disease and healthy individuals (D). The AUC values and confidence intervals are indicated.</p

Correlation of serum levels of miR-625* with the histological type of carcinoma and smoking behavior.

<p>The box plots show the different, relative amounts of miR-625* in healthy controls with unknown smoking behavior (H, n = 30) and all NSCLC patients with ADC (n = 39), SQCLC (n = 35), LCLC (n = 12), and non-smoking (NS, n = 7) and smoking (S, n = 28) behavior (A), in smoking NSCLC patients (S, n = 28) with ADC (n = 12), SQCLC (n = 10) and LCLC (n = 6) (B), and in healthy controls (H, n = 30), NS (n = 7) and S (n = 28) with malignant lung tumors (mal.), NS (n = 9) and S (n = 11) with benign lung tumors (ben.), NS (n = 16) and S (n = 39) with malignant or benign lung tumors (mal.+ben.) (C). As determined by Mann and Whitney-U test, the significant p values of the statistical evaluations of serum RNA and miR levels are indicated above the blots.</p

Differentially regulated microRNAs in healthy individuals versus patients with NSCLC.

<p>Differentially regulated microRNAs in healthy individuals versus patients with NSCLC.</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