Three-year Research Project: Circulating Tumour Cells in Women with Operable Breast Cancer

Project location: ITALY, Milan
Project start date: July 2009 - Project end date: December 2011
Project number: 2009-13

Scientific Report - Year 2
(01/01/2010 - 31/12/2010)

The vast majority of cancer-related deaths are due to metastatic, disseminated diseases. The cornerstone of this process is the generation of circulating tumor cells (CTCs) spreading from the primary tumor, colonizing distant organs and eventually causing an overtly metastatic and incurable disease.
Previous studies have shown that CTCs can be detected and isolated from the peripheral blood of women with a diagnosis of breast malignancy, including women without current evidence of disease (Meng S, et al. Clin Cancer Res 2004). In recent years the prognostic impact of CTC detection has been evaluated by different authors: nearly all the studies conclude that when CTCs are detected in patients with both localized or metastatic breast cancer they identify a group of patients with worse outcome. The milestone study in this field is the one by Cristofanilli et al. (N Engl J Med 2004) and we confirmed these results in a group of 80 metastatic breast cancer patient (Nolè et al. Ann Oncol. 2008).
Apart from detection or counting of CTCs, one of the most interesting and possibly challenging application will be the phenotypical and molecular characterization of CTC, which will allow to obtain more detailed information on CTC biology. In this regard the evaluation of CTCs could be considered as a real-time biopsy allowing the detection of possible changes in tumor phenotype. For this reason, we have verified the possibility of identifying and characterizing CTC for HER2 over-expression and the presence of stemnees and epithelial to mesenchimal phenotype markers in patient with locally advanced breast cancer, before primary or neoadjuvant preoperative chemotherapy and at the end of the treatment, before surgery, in order to provide new information to be used in the therapy of the patients.
In fact it has been shown that expression of clinically and therapeutically relevant markers such as
Estrogen Receptor (ER), Progesterone Receptor (PgR) and Human Epidermal Grow Factor Receptor 2 (HER2) can differ between a primary tumour and its relative metastasis. Gaining or losing some markers may lead to a change of the following therapy. Information on the HER2 status of CTC might be helpful for stratification and monitoring of HER2-directed therapies.
Moreover, in the last years fundamental notions of the existence of a transformed population of cells with many of the properties of stem cells that may be responsible for the origin and maintenance of tumours has gained much attention. It is thought that these cancer stem cells may play an important role in cancer establishment, progression, and resistance to current treatments. Different markers have been investigated for their ability to identify cancer stem cells, such as aldehyde dehydrogenase 1 (ALDH1), a detoxifying enzyme responsible for the oxidation of intracellular aldehydes. Although ALDH1-positive cells represented an average of only 5% of cells in tumors expressing ALDH1, ALDH1 staining positivity was associated with high histological grade and poor clinical outcome.
A number of studies have shown that carcinoma cells often activate a transdifferentiation program, termed epithelial-mesenchymal transition (EMT), to acquire the traits needed to execute the multiple steps of metastasis. Through the EMT process, epithelial cells lose cell-cell contacts and cell polarity, downregulate epithelial associated genes, acquire mesenchymal gene expression, and undergo major changes in their cytoskeleton. This cellular process culminates in a mesenchymal appearance and increased motility and invasiveness. These findings suggest that the expression of epithelial-cell surface markers, such as EpCAM, may not be optimal for detecting a heterogeneous population of CTCs including those with a mesenchymal phenotype.
We assessed the presence of CTCs and HER-2 status by the CellSearch System® and the presence of stemness and EMT markers by AdnaTest TumorStemCell and AdnaTest EMT method (AdnaGen®).
1. CTCs Quantification and Characterization using the CellSearch® system:
CellSearch System Veridex, is a standardized and semi-automated system used in our laboratory from some years for enumerating CTCs in peripheral blood. This method is based on immunomagnetic capture and enrichment of CTCs by a ferrofluid reagent consisting of nanoparticles with a magnetic core surrounded by a polimeric layer coated with antibodies targeting the EpCAM antigen for capturing CTC. After immunomagnetic capture and enrichment fluorescent reagents are added for identification and enumeration of CTCs. Besides cell quantification, the CellSearch System also allows phenotypic characterization of CTCs in terms of expression of the receptor for HER2. The CellSearch Tumor Phenotyping Reagent HER-2/neu is added to identify those CTC over-expressing HER2-neu. The identification and enumeration of CTCs were performed using the CellSpotter Analyzer, a semiautomated fluorescence-based microscopy system that permits computergenerated reconstruction of cellular images. CTCs were defined as nucleated cells lacking surface expression of the leukocyte antigen CD45 but expressing cytoplasmic cytokeratin (CK)-8, -18, or -19. An event was clas­sified as a tumor cell expressing HER2 if its morphologic features were consistent with that of a tumor cell and it exhibited the phe­notype EpCAM+, CK+, DAPI+, CD45-, and HER2/neu+.
2. Characterization of CTC by AdnaTest TumorStemCell and AdnaTest EMT method (AdnaGen®).
This CTC detection kit is available to detect the expression of stemness and EMT markers on tumor cells eventually present in five ml of peripheral blood by semiquantitative RT-PCR.
Samples were enriched for epithelial cells with anti-EpCAM coated magnetic beads. The labelled cells were extracted by a magnetic particle concentrator and subsequently lysed to extract mRNA.
The mRNA was then transcribed into cDNA which was then used as a template for tumor cell detection and characterization by multiplex PCR. The different primer mix were be used to determine breast cancer associated genes (MUC-1, HER2, and GA733-2), EMT related genes (Akt-2, Twist 1, PI3Ka) and ALDH1 which is accepted as tumor-stem cell marker. The housekeeping gene β-actin was used as internal PCR control.
Samples were considered positive if the PCR product expressed transcripts of at least one of the tumor associated genes.
1. Feasibility and reliability of HER2 determination by CellSearch System
Currently, there is no standardized and widely accepted method available for the determination of HER2 status on CTC. So we verified the feasibility and reliability of HER2 determination on cells using scraped cells obtained from primary breast tumor and spiked in blood from healthy subjects.
We have processed 36 samples with CellSearch System with different grade of HER2 overexpression and we have evaluated the concordance of HER2 status determined on primary breast tumor by immunohistochemistry (IHC) and on scraped cell from the same tumor using the CellSearch System. Moreover we have evaluated the concordance of HER2 expression determinated by FISH analysis and by CellSearch on the same scraped cells.
The results of the FISH analysis performed on the cells aspirated from the cartridge demonstrated a 100% concordance with the FISH performed on fresh tissue (22 not amplified, 9 amplified and 2 NE). Finally, different number of HER2+ scraped cells were found in 22 out of the 36 samples: 15/16 negative samples were both IHC negative and FISH not amplified, while 1/16 was IHC negative and FISH amplified in situ, with scraped cells overspressing HER2 by CellSearch processing.
The evaluation of HER2 expression on scraped cells by CellSearch System and by IHC on the corresponding tumor showed that the CellSearch method is reliable in identifying HER2 overexpression, as in all the 3+ tumors it was possible to detect variable percentage of scraped cells overexpressing HER2.
HER2 expression may be evaluated with the CellSearch System and it may be used as a preliminary method to indicate possibly HER2 positive samples which may be confirmed by FISH analysis.
2. CTCs Quantification and Characterization in locally advanced breast cancer patient
In the last year we have processed 11 samples from 9 patients (for two of these we have evaluated a baseline sample, before initiating chemotherapy, and follow up sample at the end of chemotherapy but before surgery).
For CTC enumeration by CellSearch we analyzed 22.5 ml of peripheral blood. At the baseline, 5 of the 9 (55.6%) patients had a detectable CTC count by CellSearch (range: 1-89 per 22.5 mL of PB), while in the two samples before surgery, no CTCs were detected.
Concordant HER2 expression on CTC and corresponding primary tumors was found in 3 of 5 CTC-positive patients (60%): HER2-overexpressing CTC were observed in 2 CTC-positive patients with HER2-positive primary tumors and HER2 negative CTC were detected in one patient with HER2-negative primary, confirmed by FISH.
Discordant HER2 expression was found in 2 patients with HER2-positive primary tumors in which HER2 negative CTC were detected (1 CTC/22.5 ml and 2 CTC/22.5 ml respectively).
In the AdnaTest, blood samples were regarded as CTC positive if a PCR fragment of at least one of the three markers, GA733-2, MUC-1, or HER2, was detected at a concentration of 0.15 ng/µl or greater.
In no samples tumor-stem cell and/or EMT markers were detected. At baseline, only one sample (11.1%) was positive for at least one tumor-associated gene transcripts, in particular was positive for two markers (HER2 and MUC-1), and 1 CTC/22.5 ml was detected by CellSearch. Moreover three samples were negative because no markers were detected and the remaining five baseline samples and the two samples before surgery were invalid for technical hitch occurred during the analysis.
Because of the analytical problems due to sensibility and reproducibility of the AdnaTest, the research group will try to improve the method and will proceed with patient enrolment.




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