Circulating tumor cells: what we know, what do we want to know about them and are they ready to be used in clinics?

Circulating tumor cells (CTC) present in peripheral blood are assigned precursors of advanced tumor disease. Simplicity of blood withdrawal procedure adds practically an unlimited possibility of the CTC-monitoring and the advantages of the repeated biopsies over time. CTC got prognostic, predictive and diagnostic status with the technologic advance. Although the clinical utility of CTC has reached the high evidence, the significance of CTC testing was presented in the treatment strategy mostly with palliative intention. We report on the experiences with the CTC-testing in the CLIA-like laboratory working with the size-based CTC separation and in vitro culture. The data is presented in the form of case reports in patients with breast (BC), colorectal (CRC), prostate (PC) and lung cancer (NSCLC) to support the clinical utility of CTC during the neoadjuvant, adjuvant and palliative treatment. The presented findings support the evidence for liquid biopsy clinical implementation and enhance the ability of malignant disease monitoring and the treatment efficacy prediction.

Circulating tumor cells in different stages of colorectal cancer.

INTRODUCTION: Liquid biopsies are noninvasive tests using blood or body fluids to detect circulating tumor cells (CTCs) or the products of tumor cells, such as fragments of nucleic acids or proteins that are shed into biological fluids from primary tumor or its metastates. The analysis of published clinical studies provides coherent evidence that the presence of CTCs detected in peripheral blood is a strong prognostic factor in patients with colorectal carcinoma (CRC). The aim of the study was to implement size-based separation protocol of CTCs in CRC patients. MATERIAL AND METHODS: Patients diagnosed with different stages of CRC (n = 98) were included in the study. All patients have been diagnosed for colorectal adenocarcinoma by pathology examination, 45 patients with colon carcinoma and 53 with rectosigmoid cancer. A size-based separation method (MetaCell®) for viable CTC enrichment from peripheral blood was used to assess the presence of CTCs by cytomorphological evaluation using vital fluorescence microscopy. RESULTS: Cytomorphological analysis revealed that 81 (83%) tested samples were CTC-positive and 17 (17%) were CTC-negative. We report a successful isolation of CTCs with proliferation potential in patients with CRC. The CTCs were cultured in vitro for further downstream applications. Some of the isolated CTCs were able to grow in vitro for 6 months as a standard cell culture. CONCLUSIONS: We established a reliable, inexpensive and relatively fast protocol for CTCs enrichment in CRC patients by means of vital fluorescence staining which enables their further analysis in vitro.

Molecular characterization of circulating tumor cells in ovarian cancer.

The main focus of the study was to detect circulating tumor cells (CTCs) in ovarian cancer (OC) patients using a new methodological approach (MetaCell(TM)) which is based on size-dependent separation of CTCs and subsequent cytomorphological evaluation. Cytomorphological evaluation using vital fluorescence microscopy approach enables to use the captured cells for further RNA/DNA analysis. The cytomorphological analysis is then completed by gene expression analysis (GEA). GEA showed that relative expression of EPCAM is elevated in CTC-enriched fractions in comparison to the whole peripheral blood sample and that the expression grows with in vitro cultivation time. Comparison of the relative gene expression level in the group of peripheral blood samples and CTC-fraction samples confirmed a statistically significant difference for the following genes (p < 0.02): KRT7, WT1, EPCAM, MUC16, MUC1, KRT18 and KRT19. Thus, we suggest that the combination of the above listed genes could confirm CTCs presence in OC patients with higher specificity than when GEA tests are performed for one marker only. The GEA revealed two separate clusters identifying patients with or without CTCs.

The added value of circulating tumor cells examination in ovarian cancer staging.

Delayed diagnosis of ovarian cancer (OC) is usually a cause of its high mortality. OC counts for one of the most aggressive gynecological malignancies. Noninvasive biomarkers may be used to help with diagnostic and treatment decisions in OC management. The incidence and clinical significance of occult OC cells (circulating tumor cells-CTCs) in the peripheral blood of patients with newly diagnosed or nondiagnosed OC at the time of surgical intervention were examined in our study. The objective of the study was to isolate and cultivate CTCs in OC patients (mainly stage IIIB-C) by a recently introduced size-based separation method (MetaCell(®)). CTCs were successfully isolated in patients with OC capturing cells with proliferation potential. The cells were enriched in good fitness, which enabled the short term in vitro culture of the CTCs. The CTCs may be used for further downstream applications (e.g. gene expression analysis) even if in the majority of the in vitro CTC cultures no confluence was reached. The CTCs were detected in 77 out of 118 patients (65.2%). CTC positivity was given to the relationship with different disease stage parameters with special focus on CA125 marker levels. The results show that the information on CTC presence may provide new and independent prognosis staging information to the patient description. Several interesting relationships of CA125, age and ascites presence are reported. As shown in our patient sample, patients with ascites tend to have higher CA125 levels, even if the CTCs were not found in the peripheral blood. It suggests that hematogenous dissemination is fully represented by the CTCs while lymphogenic dissemination is represented by elevated CA125. In this context, easy access to CTCs provided by the method applied in our study, both at the time of diagnosis and relapse, may become an increasingly valuable tool in future. This methodology may provide an opportunity for more personalized medicine where treatment for OC may be guided by information from an individual's CTC molecular profile.