STAGE 1/2018
Abstract Within stage 1/2018, the Ethical approval for the implementation of the study was obtained. Each patient included in the study has a unique code, also found on the set of records. The code allows the identification in the biobank of clinical, histopathological data and biological materials but does not link to its personal identification data thus respecting national and international ethical norms. Both the examination protocols and the database fields were established, as well as the workflow for the implementation and execution of the activities within subprojects 1 and 2. Also, the necessary protocols were optimized for the optimal development of the project activities such as:
1) protocol for isolating tumor cells from the peritoneum / ascites
2) isolation of total RNA from frozen tissue samples
3) RNA isolation from blood samples / tissue samples / ascites
4) miRNA analysis methodology
5) methodology for validating microRNA species by Real-Time PCR
6) cytotoxicity study methods on A2780 and SKOV-3 ovarian cancer cell lines
STAGE 2/2019
Abstract Stage 2/2019 included 19 patients with breast cancer and 54 patients with serous ovarian adenocarcinoma. Samples from patients with primary breast and ovarian tumors were banked, namely blood tumor tissue, non-tumor tissue, metastatic tissue and ascites. Tumor epithelial cells from ascites formed in serous ovarian carcinoma but also from peritoneal nodules (peritoneal carcinomatosis, CP) play an essential role in disease recurrence and drug resistance, therefore, establishing primary cultures of ascites-derived cells will allow us to develop new therapeutic strategies in the management of patients with recurrent serous ovarian cancer. Primary cultures were generated for 14 ascites samples and 10 peritoneal nodules. Genomic DNA and total RNA were isolated for further studies. The diagnostic miRNA profile that will be validated in this study will be generated following miRNA analysis on a panel of ovarian cancer-associated miRNAs as potential diagnostic targets and prognostic indicators.
Cytotoxicity experiments were initiated on the A2780 and SKOV3 cell lines. Treatments that use specific agents that target only one pathway usually fail to treat cancer. Combination treatments using chemotherapeutic agents with distinct molecular mechanisms are considered more promising for greater efficacy. Thus, during the reporting period, the optimal doses in individual treatments for 3 drugs were optimized and validated, respectively paclitaxel, cisplatin and doxorubicin, and in the next stage, combinations between the 3 chemotherapeutics with and without PARP inhibitor (AZD2461) will be tested. Were also performed validation tests for the selection of the optimal therapy and on the samples from patients with advanced ovarian cancer who show BRCA1/2 mutation and who received maintenance therapy with PARP inhibitor in the first line.
STAGE 3/2020
Stage 3/2020 included 3 breast cancer patients and 8 patients with serous ovarian adenocarcinoma. Samples from patients with primary breast and ovarian tumors were banked, as follows: blood tumor tissue, non-tumor tissue, metastatic tissue and ascites. Primary cultures were generated for the 8 samples of ascites and peritoneal nodules. The total of the samples banked by ICF is 83 (total project 1 and 2). Genomic DNA and total RNA were isolated for further studies. 8 patients from whom total RNA was isolated were included in the discovery group. These were analyzed from the point of view of the expression of a panel of genes involved in EMT: Cytokeratin 18 (CK18), Cytokeratin 19 (CK19), Vimentin, N-Caderin, E-Caderin and EpCAM and in terms of protein expression of the same markers, by immunohistochemistry technique. Immunohistochemical characterization of primary cultures derived from ascites fluid and peritoneal nodules was performed for specific epithelial markers E-Caderin and CK7, for the mesenchymal marker Vimentina, and for metabolic markers CK 8-18 / CK19 and PDL 1 – immunogenic marker. In addition, experiments evaluating a set of microRNA molecules initiated in stage 2/2019 continued with the analysis of microRNA species by microarray technology (Agilent) to identify expression profiles for ascites (AS primary culture) and peritoneal node (NP culture primary). The comparison of the expression profiles was performed by reference to the tissue peritoneal nodule, and the tumor and normal tissue, respectively. 24 samples were analyzed by microarray technology (Agilent), respectively the 6 patients from the discovery group confirmed with ovarian carcinoma. The Microarray experiment was performed using the miRNA Microarray System kit with miRNA Complete Labeling and Hyb from Agilent Technologies. The working steps were previously described and included: (1) fluorescent binding of miRNA (2) hybridization to DNA (3) blade washing and scanning (4) data processing using Agilent 12.0.3.1 software (contains 3000 human species of miRNA). Bioinformatics analysis of expression signatures of microRNA species revealed a top 10 statistically significantly modified miRNA (overexpressed or underexpressed) between primary cell cultures isolated from (AS) and peritoneal node (NP) in direct comparison with NP tissue and tumor tissue (T) / normal (N). Preliminary data obtained for the 6 patients included in the discovery group allowed the selection of an miRNA panel for validation by RT-PCR on the validation group. The selection was based on their different expression in the primary culture (AS and NP) compared to NP tissue or tumor tissue (overexpressed or under-expressed) and ensuring from the beginning that we have a sufficiently high level of expression given of Agilent microarray platform. The diagnostic miRNA profile that will be validated in this study will be generated following miRNA analysis on a panel of ovarian cancer-associated miRNAs as potential diagnostic targets and prognostic indicators.
The experiments on the A2780 and SKOV3 cell lines continued in stage 3/2020 through the functional evaluation of some drugs such as: paclitaxel (PCX), doxorubicin (DOX), cisplatin (CPT) and AZD2461 (PARP inhibitor, new generation agent after olaparib). To determine the concentration of each drug to which the response is reduced by half, viability was determined and IC50 was calculated using the MTT technique. Additionally, the migration rate of SKOV3 cells was evaluated in response to selected CPT and DOX doses. The degree of healing of the label created provides information on both the characteristics of migration, cell viability and the ability to interact with other cells, as well as additional data on how the tested molecules can affect each of these parameters. At the end of the experiment, the cells were collected for RNA and protein isolation. RNA is used to analyze changes developed based on the EMT phenomenon, as a result of treatment with selected drug doses (as single agents and in combination with AZD2461) and proteins will be analyzed by Luminex multiplex technique. Cell migration is an integral part of basic biological processes, such as tissue formation, the repair process and the immune response, as well as the processes of tumor invasion and metastasis. Advanced knowledge and understanding of the mechanism of collective cell migration is crucial to develop new therapeutic strategies.