1. Complementary detection strategies for circulating tumor cells in breast cancer: clinical implications of combining immunofluorescence and cytopathological stainingTanja Jesenko, Cvetka Grašič-Kuhar, Živa Pišljar, Simona Miceska, Veronika Kloboves-Prevodnik, Maja Čemažar, 2025, izvirni znanstveni članek Povzetek: Background: Circulating Tumor Cells (CTCs) serve as important biomarkers for disease monitoring and treatment response in patients with metastatic breast cancer. Their detection remains challenging because of their low abundance, phenotypic diversity and non-standardized mode of detection. Cytopathological Giemsa and Immunofluorescence (IF) staining can offer complementary approaches for CTC characterization. Giemsa staining enables assessment of cellular morphology, while IF allows for marker-specific identification, together providing a more comprehensive and accurate evaluation of CTCs. Methods: We developed an IF staining protocol with antibodies against Cytokeratin (CK), vimentin (VIM), and Cluster of Differentiation 45 (CD45) to distinguish epithelial, mesenchymal, hybrid and hematopoietic cells for CTC detection and characterization and compared it with cytopathologic method of detection via Giemsa staining with regard to CTC detection rates and morphological detail. Results: Study was performed on the samples of 29 heavily pretreated patients with metastatic breast cancer (median duration of metastatic disease 19.4 months). Giemsa staining enabled the detection of a higher number of CTCs compared to our IF protocol. Lower detection rate was potentially due to the loss of fragile or loosely adherent cells during methanol fixation and IF staining. Additionally, in IF-stained samples, some CTCs presented faint nuclear signals, potentially impairing their recognition. The IF staining supported the identity of CTCs detected on Giemsa-stained slides by employing a three-color antibody panel-based approach and allowed detailed phenotypic discrimination and structural analysis of CTCs, including the identification of a distinctive CK polarization pattern suggestive of a transitional state during intravasation. Conclusion: Giemsa and IF may thus be complementary rather than mutually exclusive and relying on a single detection approach could underestimate the true CTC burden. An integrative strategy combining both techniques may offer a more comprehensive view of CTC populations in metastatic breast cancer, thereby enhancing diagnostic precision.
Ključne besede: biomarkers, breast cancer, circulating tumor cells, cytopathological detection
Objavljeno v DiRROS: 05.12.2025; Ogledov: 1002; Prenosov: 163
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2. Morphological features of breast cancer circulating tumor cells in blood after physical and biological type of isolationTanja Jesenko, Živa Pišljar, Cvetka Grašič-Kuhar, Maja Čemažar, Urška Matkovič, Simona Miceska, Jerneja Varl, Anamarija Kuhar, Veronika Kloboves-Prevodnik, 2021, izvirni znanstveni članek Povzetek: Background. Circulating tumor cells (CTCs) have become an important biomarker in breast cancer. Different iso-lation tech-niques based on their biological or physical features were established. Currently, the most widely used methods for visualization after their separation are based on immunofluorescent staining, which does not provide the information on the morphology.Materials and methods. The aim of this study was to evaluate how two different separation techniques affect cell morphology and to analyse cell morphology with techniques used in routine cytopathological laboratory. A direct side-by-side comparison of physical (Parsortix%) and biological (MACS%) separation technique was performed.Results. In the preclinical setting, both isolation techniques retained the viability and antigenic characteristics of MCF7 breast cancer cells. Some signs of degeneration such as cell swelling, cytoplasmic blebs, villous projections and vacuolization were observed. In metastatic breast cancer patient cohort, morphological features of isolated CTCs were dependent on the separation technique. After physical separation, CTCs with preserved cell morphology were detected. After biological separation the majority of the isolated CTCs were so degenerated that their identity was difficult to confirm.Conclusions. Taken together, physical separation is a suitable technique for detection of CTCs with preserved cell morphology for the use in a routine cytopathological laboratory.
Ključne besede: circulating tumor cells, breast cancer, morphology
Objavljeno v DiRROS: 22.07.2024; Ogledov: 1372; Prenosov: 884
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