Morfologická a genomická charakterizace cirkulujících nádorových buněk u metastatického kolorektálního karcinomu

Colorectal cancer (CRC) is the third most common cancer worldwide; it is responsible for nearly 10% of all newly diagnosed cancers and is the second most cause of cancer related death in Europe. Biomarkers for therapy guidance, targeted therapy and survival prognosis are still limited. As CRC is a heterogeneous disease, different parts of the tumor might have varying molecular characteristics which may change during therapy or disease progression. Through solid biopsies and screenings, these local or temporal differences are impossible to monitor. To facilitate detection of these possible temporal changes, a regularly and non-invasively accessible biomarker is required for disease monitoring. Circulating tumor cells (CTCs) might represent such a biomarker as they have been shown to be fluid surrogates of the solid tumor. EpCAM positive CTCs have shown to be prognostic in CRC for survival, but their full potential has not yet been evaluated further. By using the High Definition Single Cell Analysis (HD-SCA) workflow, we were able to analyze the entire spectrum of CTCs and categorize them as the regular CTCs (HD-CTC), CTCs with a smaller nuclear area (CTC-Small), CTCs with low expression of epithelial marker cytokeratin (CTC-LowCK) and CTCs undergoing apoptosis and therefore releasing cell free DNA...Kolorektální karcinom (CRC) je celosvětově třetím nejčastějším nádorovým onemocněním - je odpovědný za téměř 10 % všech nově diagnostikovaných nádorů a je druhou nejčastější příčinou úmrtí spojeného s nádory v Evropě. Paleta biomarkerů použitelných pro výběr vhodné terapie, cílené léčby a pro prognózu přežívání je stále omezená. Vzhledem k tomu, že CRC je heterogenní onemocnění, různé části nádoru mohou mít odlišné molekulární charakteristiky, které se mohou měnit během léčby nebo progrese onemocnění. Tyto místní nebo časové rozdíly není možné efektivně monitorovat prostřednictvím klasické biopsie. Pro umožnění detekce nádorové evoluce je potřebný neinvazivní a opakovaně dostupný biomarker. Tento biomarker mohou představovat cirkulující nádorové buňky (CTC), jelikož reprezentují solidní nádor v krevním řečišti. Bylo prokázáno, že EpCAM pozitivní CTC mají prognostický efekt u pacientů s CRC, ale jejich plný potenciál nebyl dosud prozkoumán. S využitím metody HD-SCA jsme mohli analyzovat celé spektrum CTC a klasifikovat je jako běžné CTC (HD-CTC), CTC s menším jádrem (CTC-Small), CTC s nízkou expresí epiteliálního znaku cytokeratinu (CTC-LowCK) a CTC podstupující apoptózu a tedy uvolňující DNA (CTC-cfDNA produkující). Navíc jsme detekovali a analyzovali CTC clustery (CTCC). Analýza zahrnovala nejen...Ústav histologie a embryologieLékařská fakulta v PlzniFaculty of Medicine in Pilse

Morphological and Genomic Profiling of Circulating Tumor Cells in Metastatic Colorectal Cancer

Colorectal cancer (CRC) is the third most common cancer worldwide; it is responsible for nearly 10% of all newly diagnosed cancers and is the second most cause of cancer related death in Europe. Biomarkers for therapy guidance, targeted therapy and survival prognosis are still limited. As CRC is a heterogeneous disease, different parts of the tumor might have varying molecular characteristics which may change during therapy or disease progression. Through solid biopsies and screenings, these local or temporal differences are impossible to monitor. To facilitate detection of these possible temporal changes, a regularly and non-invasively accessible biomarker is required for disease monitoring. Circulating tumor cells (CTCs) might represent such a biomarker as they have been shown to be fluid surrogates of the solid tumor. EpCAM positive CTCs have shown to be prognostic in CRC for survival, but their full potential has not yet been evaluated further. By using the High Definition Single Cell Analysis (HD-SCA) workflow, we were able to analyze the entire spectrum of CTCs and categorize them as the regular CTCs (HD-CTC), CTCs with a smaller nuclear area (CTC-Small), CTCs with low expression of epithelial marker cytokeratin (CTC-LowCK) and CTCs undergoing apoptosis and therefore releasing cell free DNA..

lncRNAs in Non-Malignant Tissue Have Prognostic Value in Colorectal Cancer

Although colorectal cancer (CRC) is the third most frequent cause of cancer related death in Europe, clinically relevant biomarkers for therapy guidance and prognosis are insufficiently reliable. Long non-coding RNAs (lncRNAs) are RNAs over 200 nucleotides long that are not translated into proteins but can influence biological processes. There is emerging evidence for their involvement in solid cancer as oncogenes, tumour suppressors or regulators of cell proliferation and metastasis development. The goal of this study was to evaluate the prognostic effect of selected lncRNAs in a retrospective study on CRC patients from the Czech Republic. We used a quantitative PCR approach to measure the expression in paired non-malignant and tumour tissue samples of CRC patients of nine lncRNAs previously shown to be involved in cancer progression—ANRIL, CCAT1, GAS5, linc-ROR, MALAT1, MIR155HG, PCAT1, SPRY4-IT1 and TUG1. Associations between expression and expression ratios and clinical characteristics and survival were assessed by using univariable Cox proportional hazards models, Kaplan-Meier estimations with the Gehan-Wilcoxon test, the Mann-Whitney U test, the Kruskal-Wallis test and Spearman’s correlations. A comparison of expression in tumour tissue (TT) and non-malignant mucosa tissue (MT) showed significant upregulation of CCAT1 and linc-ROR in TT (p < 0.001 and p = 0.001, respectively) and downregulation of ANRIL, MIR155HG and MALAT1 (p = 0.001, p = 0.010, p = 0.001, respectively). Linc-ROR was significantly associated with the presence of synchronous metastases (p = 0.033). For individual tissue types, lower MIR155HG expression in TT was correlated with both shorter overall survival (p = 0.008) and shorter disease-free survival (p = 0.040). In MT, expression ratios of CCAT1/ANRIL and CCAT1/MIR155HG were associated with overall survival (p = 0.005 and p = 0.006, respectively). Our results revealed that changes in expression of lncRNAs between MT and TT hold potential to be used as prognostic biomarkers in CRC patients. Moreover, the ratios of CCAT1 to ANRIL and MIR155HG in MT also exhibit potential for prognosis assessment without tumour sampling. Our results also indicate that cancer progression is associated with detrimental system-wide changes in patient tissue, which might govern patient survival even after successful elimination of tumour or cancerous cells

Circulating Tumor Cell Kinetics and Morphology from the Liquid Biopsy Predict Disease Progression in Patients with Metastatic Colorectal Cancer Following Resection

The liquid biopsy has the potential to improve current clinical practice in oncology byproviding real-time personalized information about a patient’s disease status and response to treatment. In this study, we evaluated 161 peripheral blood (PB) samples that were collected aroundsurgical resection from 47 metastatic colorectal cancer (mCRC) patients using the High-DefinitionSingle Cell Assay (HDSCA) workflow. In conjunction with the standard circulating tumor cell (CTC)enumeration, cellular morphology and kinetics between time-points of collection were considered inthe survival analysis. CTCs, CTC-Apoptotic, and CTC clusters were found to indicate poor survivalwith an increase in cell count from pre-resection to post-resection. This study demonstrates thatCTC subcategorization based on morphological differences leads to nuanced results between thesubtypes, emphasizing the heterogeneity within the CTC classification. Furthermore, we show thatfactoring in the time-point of each blood collection is critical, both for its static enumeration and forthe change in cell populations between draws. By integrating morphology and time-based analysisalongside standard CTC enumeration, liquid biopsy platforms can provide greater insight into thepathophysiology of mCRC by highlighting the complexity of the disease across a patient’s treatment