Liquid biopsy is a common term referring to circulating tumor cells and other biomarkers, such as circulating tumor DNA (ctDNA) or extracellular vesicles. Liquid biopsy presents a range of clinical advantages, such as the low invasiveness of the blood sample collection and continuous control of the tumor progression. In addition, this approach enables the mechanisms of drug resistance to be determined in various methods of cancer treatment, including immunotherapy. However, in the case of melanoma, the application of liquid biopsy in patient stratification and therapy needs further investigation. This review attempts to collect all of the relevant and recent information about circulating melanoma cells (CMCs) related to the context of malignant melanoma and immunotherapy. Furthermore, the biology of liquid biopsy analytes, including CMCs, ctDNA, mRNA and exosomes, as well as techniques for their detection and isolation, are also described. The available data support the notion that thoughtful selection of biomarkers and technologies for their detection can contribute to the development of precision medicine by increasing the efficacy of cancer diagnostics and treatment.

The treatment of non-small cell lung cancer (NSCLC) has recently evolved with the introduction of targeted therapy based on the use of tyrosine kinase inhibitors (TKIs) in patients with certain gene alterations, including EGFR, ALK, ROS1, BRAF, and MET genes. Molecular targeted therapy based on TKIs has improved clinical outcomes in a large number of NSCLC patients with advanced disease, enabling significantly longer progression-free survival (PFS). Liquid biopsy is an increasingly popular diagnostic tool for treating TKI-based NSCLC. The studies presented in this article show that detection and analysis based on liquid biopsy elements such as circulating tumor cells (CTCs), cell-free DNA (cfDNA), exosomes, and/or tumor-educated platelets (TEPs) can contribute to the appropriate selection and monitoring of targeted therapy in NSCLC patients as complementary to invasive tissue biopsy. The detection of these elements, combined with their molecular analysis (using, e.g., digital PCR (dPCR), next generation sequencing (NGS), shallow whole genome sequencing (sWGS)), enables the detection of mutations, which are required for the TKI treatment. Despite such promising results obtained by many research teams, it is still necessary to carry out prospective studies on a larger group of patients in order to validate these methods before their application in clinical practice.

Despite the rising public awareness of the risk factors and the possible prevention of melanoma development, it remains challenging in terms of diagnosis and treatment. To improve the clinical situation of patients, it would be especially beneficial to develop prognostic methods for the effective and continuous assessment of the disease course. The solution could lie in the selection of effective biomarkers derived from the tumor microenvironment, increasing the effectiveness of melanoma prognoses and monitoring. Hence, in this study, we evaluated the number of circulating melanoma cells (CMCs) in representative blood samples of melanoma patients vs. healthy controls, as well as the proportion of particular cytotoxic T cells in the total lymphocyte and leukocyte population as a reflection of immune resistance. The results were correlated with the clinical parameters of the patients to examine the potential value of CMC quantification and lymphoid cell phenotyping in melanoma diagnostics, prognostics, and treatment outcome monitoring. The CMC numbers were significantly higher in melanoma patients than in healthy controls. However, an analysis of the correlations between the baseline CMC counts and the clinical parameters found no significant results. In turn, we found significant differences between the groups in the percentage of various profiles of CD8+ cytotoxic T lymphocytes characterized by TIGIT and TIM-3 differential expression. Importantly, the CMC number correlated with CD8+TIGIT+ and CD8+TIGIT+TIM-3- cytotoxic T cell counts in the melanoma patient group. Considering the above, the combination of CMCs and the immunological status of the patient, as defined by the prevalence of selected immune cell types, seems to be a promising approach in melanoma diagnostics and prognostics.

Immobilization of antibodies has a number of promising applications, including detection of biomolecules and cells. Well-oriented antibodies are required to bind them effectively. To eliminate the problem of random antibodies’ orientation, the surface of the device can be modified with silanes. This study aimed at elucidating if selected aminosilanes were able to bind antibodies in the appropriate orientation and thus retain their binding activity. Silanization of glass slides was performed using three amino-functional trialkoxysilanes – A, AE, and AEE. The immunofluorescent reaction was used to evaluate the potential of the silanized glass surface to bind anti-EpCAM antibodies. The affinity of selected anti-EpCAM HEA125 antibodies labeled with fluorochrome to tested silanized surfaces was evaluated by measuring the mean fluorescence intensity (MFI) in each analyzed area. The presented silanes effectively bound antibodies. Higher fluorescence intensity was noticed in the case of silane-coated glass slides in comparison to unmodified ones. The differences in the contact angles also confirmed this result. In the case of silane A, the fluorescence intensity reflected the amount of bound antibodies. However, there was no such a relation in the case of the silanes AE and AEE. Although our research gave promising results, the usefulness of selected silanes needs to be confirmed by further studies using cancer cells. Running title: Aminosilanes as enhancers of antibody immobilization

This article provides a historical overview of melanoma, involving the knowledge of this neoplasm from antiquity to the present. Selected people who made key descriptions of the disease, its symptoms, and treatment methods were listed. The classification of melanoma, which is used in therapeutic management nowadays, is briefly discussed. Additionally, we describe circulating tumour cells and the selected diagnostic methods associated with their detection and characteristics. The aim of this article is to present a historical outline of melanoma, as well as its classification and the development of laboratory methods of its diagnosis. In addition, we have also provided a comparison of historical and current knowledge of this malignancy.