Dissertation committee:
Βασίλειος Σμυρνιώτης, Καθηγητής Χειρουργικής Ιατρική, ΕΚΠΑ
Παρασκευή Μουτσάτσου-Λαδικού, Καθηγήτρια Κλινικής Βιοχημείας, Ιατρική, ΕΚΠΑ
Πέτρος Καρακίτσος, Καθηγητής Ιατρικής, ΕΚΠΑ
Γεώργιος Αθανασάς, Αναπληρωτής Καθηγητής Ιατρικής, ΕΚΠΑ
Κλεάνθη Δήμα, Αναπληρώτρια Καθηγήτρια Ιατρικής, ΕΚΠΑ
Νικόλαος Δανιάς, Επίκουρος Καθηγητής Ιατρικής, ΕΚΠΑ
Παύλος Μαραγκουδάκης, Επίκουρος Καθηγητής Ιατρικής, ΕΚΠΑ
Summary:
Circulating Tumor Cells (CTCs) are detected in peripheral blood of patients with epithelial origin cancers in concentrations of 1 cancer cell per 106-107 leukocytes. They represent an important biological link in the prevalence of cancer, between primary tumor and metastatic disease. The clinical significance of their detection has been studied by several groups and it has been proven that they represent an independent prognostic factor for Disease Free and Overall Survival. They are promising markers for the evaluation of response to therapy and they could be investigated as new therapeutic targets.
The so far used techniques for CTCs detection can be generally divided in cytometric techniques looking at the entire cell and the molecular techniques. Nucleic acid based techniques are mostly represented by RT-PCR whereas immunocytochemistry, Laser Scanning Cytometry, CellSearch system and Flow Cytometry are cytometric techniques.
The CTCs are very rare events, thus an isolation step sould be performed before their detection. The isolation can be based on the structural characteristics of the cells (cell size and density) or by using specific tumor markers. Immunomagnetic separation can be divided into negative selection (depletion of leukocytes) using CD45 antibody and positive selection using antibodies common to all tumor cells (EpCAM) or tumor specific antibodies.
Molecular techniques are considered as a gold standard for CTCs detection, because they are characterized by high sensitivity and specificity as well. Few reports have been published on the CTCs detection using Flow Cytometry.
The aim of the present PhD thesis was the comparison of two methods (i.e molecular and flow cytometry) for detection and enumeration of CTCs in peripheral blood of colon cancer patients. Furthermore, to investigate any correlation between the two techniques according to their sensitivity and specificity, their LOD, and finally, the determination of cut off values and the choice of the appropriate “Tumor specific” markers.
The material of the study consisted of 84 samples from peripheral blood drawals from colon cancer patients: a) before the surgery and b) the day after the surgery. The control group consisted of 16 healthy blood donors.
The methods used were molecular techniques (PCR, Nested PCR and RT-PCR), along with flow cytometry.
The study was conducted at the laboratory of Diagnostic Cytopathology, in the University General Hospital “Attikon”, in cooperation with the 4th Department of the University Surgery Clinic. The study protocol was approved by the Ethics Committee of the University General Hospital “Attikon”.
All statistical analyses were performed using statistical package SPSS statistics for Windows Version: 20.0. For independent variables the t-test was performed and for categorical variables the Fisher’s exact test. Between-group comparison was undertaken using Mann-Whitney test. Linear correlation between two variables was evaluated using Spearman correlation (rho). P˂0.05 were considered to be statistically significant.
The results are given below:
Molecular techniques
A statistically significant increase in the pathological samples was identified only in the detection of CK19 trranscripts (with p=0.024) using the nested method, while the correlation with the stage of tumor was weak (Grade, p=0.012).
A larger sensitivity (SN) was exhibited by the CK19 nested (67.6%) with specificity (SP) 66.2%. For the CK20 and the traditional PCR for CK19 the corresponding percentages were considerably lower (SN: 2.9%, 30.9%, SP: 100%, 75%). The detection of EGFR nested exhibited SN: 22.1%, SP: 71.2%.
Flow Cytometry
The most significant correlation identified was for the expression of CK with carcinomas (0.256, p=0.044), with the existence of lymph node infiltration (0.380 p=0.008) and with the stage of the disease (0.391, p=0.003). For the direct protocol, considerable correlation was found with the samples of the right colon (0.369, p=0.002) and with the positivity for nested CK19 (0.241, p=0.042).
The detection with the direct protocol exhibited, with threshold 3 cells/mL (cut off value), SN: 49.2% with SP: 58.3%, while the intracellular, with threshold 1 cell/mL (cut off value) SN: 62.7% with SP: 70%.
To conclude, the comparison of the two techniques employed in the present doctoral thesis, namely molecular (PCR, Nested PCR and RT-PCR) and flow cytometry, demonstrated the advantage of flow cytometry, without undermining the value of molecular techniques and particularly of the currently introduced and used in modern laboratories techniques of the quantitative PCR (qPCR) and of the real-time quantitative PCR [Real‐Time Quantitative PCR (qPCR].
Summarazing, CTCs can be used as an additional predictor for disease progression in newly diagnosed patients with malignant cancers and therefore are characterized as “real-time liquid biopsy”. The molecular profile of CTCs could also identify therapeutic targets such as the HER2 or EGFR for individualized therapy through the action of biological therapies with monoclonal antibodies such as Herceptin or Erbitux. Therefore, detection of CTCs in the peripheral blood could become not only an excellent non-invasive prognostic tumor marker but also an assistant in the classification of the disease and choice of therapeutic intervention.
Keywords:
Circulating tumor cells, Liquid biopsy, Detection, Flow cytometry, Molecular techniques
