Monitoring of breast cancer progression via aptamer-based detection of circulating tumor cells in clinical blood samples.

Introduction: Breast cancer (BC) diagnostics lack noninvasive methods and procedures for screening and monitoring disease dynamics. Admitted CellSearch® is used for fluid biopsy and capture of circulating tumor cells of only epithelial origin. Here we describe an RNA aptamer (MDA231) for detecting BC cells in clinical samples, including blood. The MDA231 aptamer was originally selected against triple-negative breast cancer cell line MDA-MB-231 using cell-SELEX. Methods: The aptamer structure in solution was predicted using mFold program and molecular dynamic simulations. The affinity and specificity of the evolved aptamers were evaluated by flow cytometry and laser scanning microscopy on clinical tissues from breast cancer patients. CTCs were isolated form the patients' blood using the developed method of aptamer-based magnetic separation. Breast cancer origin of CTCs was confirmed by cytological, RT-qPCR and Immunocytochemical analyses. Results: MDA231 can specifically recognize breast cancer cells in surgically resected tissues from patients with different molecular subtypes: triple-negative, Luminal A, and Luminal B, but not in benign tumors, lung cancer, glial tumor and healthy epithelial from lungs and breast. This RNA aptamer can identify cancer cells in complex cellular environments, including tumor biopsies (e.g., tumor tissues vs. margins) and clinical blood samples (e.g., circulating tumor cells). Breast cancer origin of the aptamer-based magnetically separated CTCs has been proved by immunocytochemistry and mammaglobin mRNA expression. Discussion: We suggest a simple, minimally-invasive breast cancer diagnostic method based on non-epithelial MDA231 aptamer-specific magnetic isolation of circulating tumor cells. Isolated cells are intact and can be utilized for molecular diagnostics purposes.

Bioluminescent SNP genotyping technique: Development and application for detection of melanocortin 1 receptor gene polymorphisms.

SNP genotyping based on the reaction of specific primer extension with the following bioluminescent detection of its products was shown to be potentially applicable for biomedical exploration. The paper describes its elaboration and first application in extensive biomedical research concerning MC1R gene variants' frequency and associations with clinical characteristics in melanoma patients of Eastern Siberia (Krasnoyarsk region, Russia). Polymorphisms rs 1805007 (R151C), rs 1805008 (R160W), and rs 1805009 (D294H) were detected in 174 DNA samples from patients with histologically proved diagnosis of cutaneous melanoma and in 200 samples from healthy individuals. All the results on bioluminescent SNP genotyping were confirmed by Sanger sequencing. Some features characteristic of the population were found, i.e. melanoma is mostly associated with R160W or R151C while variant D294H is extremely rare; simultaneous carriage of any two investigated variants is also strongly associated with melanoma; R151C is associated with ulceration and consequently the disease course is more aggressive, etc. The design of the technique allows fast evaluation of any known diagnostically important SNP frequencies and associations across population.