Giulia Gallerani

Detection and recovery of circulating colon cancer cells using a dielectrophoresis-based device: KRAS mutation status in pure CTCs

The characterization of circulating tumor cells (CTCs) could substantially improve the management of cancer patients. However, their study is still a matter of debate, often due to lymphocyte contamination. In the present paper, an investigation of CTCs was carried out for the first time using DEPArray, a dielectrophoresis-based platform able to detect and sort pure CTCs. Analyses were conducted on peripheral blood (PB) samples from patients with metastatic colon cancer. After 100% pure cell recovery and whole genome amplification, KRAS gene mutation of CTCs was screened and compared to gene status in the primary tumor tissue. CTCs were found in 21 colon cancer patients (52.5%), with more than three tumor cells per 7.5 ml. KRAS gene mutation analysis, showed a mutational concordance between CTCs and primary tumor in 50% of matched cases. The present study demonstrates for the first time the feasibility of analyzing at the molecular level pure CTCs avoiding lymphocyte contamination using an innovative instrumentation, and a KRAS discordance between CTCs and primary tissue. Our results present dielectrophoresis-based procedures as a new standard in single cell analysis and recovery and invite careful reflection on the value of CTCs characterization.

Epithelial Mesenchymal Transition: a double-edged sword

Epithelial mesenchymal transition (EMT) is a physiological process necessary to normal embryologic development. However in genesis of pathological situations, this transition can be perverted and signaling pathways have different regulations from those of normal physiology. In cancer invasion, such a mechanism leads to generation of circulating tumor cells. Epithelial cancer cells become motile mesenchymal cells able to shed from the primary tumor and enter in the blood circulation. This is the major part of the invasive way of cancer. EMT is also implicated in chronic diseases like fibrosis and particularly renal fibrosis. In adult organisms, healing is based on EMT which is beneficial to repair wounds even if it can sometimes exceed its goal and elicit fibrosis. In this review, we delineate the clinical significance of EMT in both physiological and pathological circumstances.

Circulating tumor cells and epithelial, mesenchymal and stemness markers: characterization of cell subpopulations

Until now detection and numeration of circulating tumor cells (CTCs) were essentially used as a prognostic factor in cancer progression. To extend the role of these kinds of analysis, it seems necessary to improve analytical methods related to isolation and characterization of CTCs. Discrepancies between published results corroborates this requirement. In this review we suggest a combination of markers able to reach the goal. Moreover to improve the clinical utility of CTC analysis, particularly in the therapeutic follow up of the disease, epithelial mesenchymal transition (EMT) level of a global CTC population should be studied.

Circulating tumor cells in early breast cancer: A connection with vascular invasion.

Although circulating tumor cells (CTCs) have been studied in early breast cancer (EBC), their value in this setting is still not fully understood. We isolated and studied CTCs in the peripheral blood (PB) of 48 EBC patients pre-surgery and one and 6 months post-surgery using an approach involving EpCAM-independent enrichment and a dielectrophoresis-based device. Method feasibility and the correlation between CTCs and primary tumor features were evaluated. CTCs were found in 27.1% of pre-surgery patients, 20.9% of patients one-month post-surgery, and about 33% of patients 6-months post-surgery. CTCs were recovered singly for further molecular characterization. Pre-surgery CTC-positive patients more frequently had negative prognostic features, i.e. high proliferation, large tumor dimension, lymph node positivity and negative receptor status than the other subgroup. In particular, vascular invasion showed a statistically significant correlation with CTC-positivity. Our procedure proved feasible and capable of recovering CTCs from EBC patients. Furthermore, our results suggest that CTCs may be linked to vascular invasion and to other known negative prognostic factors.

CTCs in early breast cancer: A path worth taking

Circulating tumor cells (CTCs) are cellular elements of undeniable significance that spread from the tumor mass into the peripheral blood and constitute one of the main vehicles for disease diffusion. Their rarity, in addition to a number of molecular and cellular features, has severely impaired research and exploitation. CTCs have been evaluated in early breast cancer (EBC), although long from being fully accepted in this field also due to a lack of technical standardization. CTCs hold promise to be a powerful non-invasive real-time measurable biomarker in all disease stages. This hypothesis is particularly appealing in the adjuvant setting of breast cancer, as it still lacks a marker that could play a central role in monitoring disease-free intervals, predicting early relapse and guiding drug selection. This review aimed to discuss CTC characteristics and show the main results of CTC-research in EBC setting, stating the urgency to continue basic and translational research in this field to definitely translate this marker from bench to bedside.

Splicing factor ratio as an index of epithelial-mesenchymal transition and tumor aggressiveness in breast cancer

Epithelial-to-mesenchymal transition (EMT) has been shown to be associated with tumor progression and metastasis. During this process in breast cancer, a crucial role is played by alternative splicing systems. To identify a new early prognostic marker of metastasis, we evaluated EMT-related gene expression in breast cell lines, and in primary tumor tissue from 31 patients with early breast cancer, focusing our attention on EMT-related splicing factors ESRP1, ESRP2 and RBFOX2. Results showed that the expression patterns of these genes were indicative of the onset of EMT in in-vitro models, but not in tissue samples. However, the ratio between ESRP1 or ESRP2 and RBFOX2 significantly decreased during EMT and positively correlated with the EMT-specific phenotype in cell models, representing a promising prognostic markers. Low ESRP1/RBFOX2 ratio value was associated with a higher risk of metastasis (p < 0.005) in early breast cancer patients, regardless other clinical features. A cut-off of ratio of 1.067 was determined by ROC curve analysis (AUC 0.8375; 95% CI 0.6963–0.9787). Our study show evidence that a decrease in this ratio correlates with cancer progression. The results provide a rationale for using ESRP1/RBFOX2 ratio as a new prognostic biomarker for the early prediction of metastatic potential in breast cancer.

Circulating Tumor Cells in the Adenocarcinoma of the Esophagus.

Circulating tumor cells (CTCs) are elements of indisputable significance as they seem to be responsible for the onset of metastasis. Despite this, research into CTCs and their clinical application have been hindered by their rarity and heterogeneity at the molecular and cellular level, and also by a lack of technical standardization. Esophageal adenocarcinoma (EAC) is a highly aggressive cancer that is often diagnosed at an advanced stage. Its incidence has increased so much in recent years that new diagnostic, prognostic and predictive biomarkers are urgently needed. Preliminary findings suggest that CTCs could represent an effective, non-invasive, real-time assessable biomarker in all stages of EAC. This review provides an overview of EAC and CTC characteristics and reports the main research results obtained on CTCs in this setting. The need to carry out further basic and translational research in this area to confirm the clinical usefulness of CTCs and to provide oncologists with a tool to improve therapeutic strategies for EAC patients was herein highlighted.

DNA ploidy and S-phase fraction analysis in peritoneal carcinomatosis from ovarian cancer: correlation with clinical pathological factors and response to chemotherapy

Objective We investigated the correlation between ploidy or S-phase fraction (SPF) and the clinical pathological characteristics of patients with peritoneal carcinomatosis from ovarian cancer. We also assessed their relation with the in vivo and in vitro response to several chemotherapeutic agents. Patients and methods Fifty-three patients with peritoneal carcinomatosis from ovarian cancer were enrolled. Frozen tumor tissue was dissociated by a detergent–trypsin method, and the resulting cell suspension was stained with RNase A and propidium iodide. Samples were then analyzed for ploidy and SPF by flow cytometry. Fresh tumor tissue was dissociated by enzymatic digestion, and cells were exposed to different concentrations of cisplatin, adriamycin, carboplatin, gemcitabine and taxol for 72 hours. In vitro drug sensitivity was then measured using the sulforhodamine B assay. Results No significant correlation was found between ploidy or SPF and patient characteristics, even though primary carcinomas were mainly hyperdiploid and more proliferative than recurrent tumors. SPF differed significantly among ploidy categories (P=0.01), and high SPF was associated with short-term survival (P=0.48). Patients with multiploid tumors were the most resistant to platinum-based chemotherapy, whereas those with hyperdiploid tumors were the most responsive. In vitro multiploid tumors were the least sensitive, while hypodiploid samples showed the highest sensitivity to the tested drugs. Sensitivity to adriamycin was significantly correlated with ploidy (P=0.03), whereas sensitivity to taxol was correlated with SPF (P=0.04). Conclusion Our results indicate that ploidy and SPF could facilitate the choice of therapy for patients with peritoneal carcinomatosis.

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

Circulating Tumor Cells: Back to the Future

Circulating tumor cells (CTCs) are rare and heterogeneous cells found in the peripheral blood of cancer patients. They are supposed to be a central component of metastatic dissemination and have been correlated with prognosis, progression-free survival, and treatment efficacy in different solid tumors (1, 2). Despite CTC significance, studies conducted on them still present considerable issues. In particular, regarding to how CTCs should be investigated and to their actual metastatic competence and tumor heterogeneity reflecting dissimilar cancer cell subpopulations. Importantly, CTCs’ clinical utility has not been demonstrated and they cannot be utilized to guide therapeutic decisions. As reviewed by others (3), recent and currently ongoing clinical trials are trying to determine the predictive role of CTCs, but they are apparently failing to support definitive conclusions. Nevertheless, the results of these trials could shed a light on the real possibilities of CTCs. In our opinion, CTC troubleshootings will depend on what scientific community will explore about CTCs and the metastatic process. Only a deeper insight in base knowledge about CTCs and their role in tumor biology may help us in overcoming such hurdles.