NEW PERSPECTIVE IN PROSTATE CANCER DIAGNOSIS AND TREATMENT: A PIVOTAL STUDY ON EEF1A1 PROTEIN LEVELS AS POTENTIAL THERAPEUTIC TARGET AND LIQUID BIOPSY ON CFDNA OF EEF1A1, CFRNA EEF1A1 AND CFDI OF LINE-1 AS NEW DIAGNOSTIC AND PROGNOSTIC BIOMARKERS

The overall aim of our project was the further development and validation in real settings of the highly sensitive and quantitative diagnostic platform digital droplet PCR for the early and fast detection of plasma cfDNA quantity and cfDI of repetitive elements, such as LINE-1, associated to prostate cancer. This non-invasive blood test could serve to better define the diagnosis, identifying high risk PCa patients. If validated, the test could enter the clinical practice, reducing the need of patient’s biopsy and, thus, health care costs and patients’ suffering. Moreover, the test could have the potential to be develop as a screening method or as a test for the molecular stratification of patients in the follow-up. Secondary endpoint: evaluate the diagnostic and prognostic value of liquid biopsy of biomarkers like eEF1A1 e eEF1A2. The rationale is that the research group has demonstrated the role of eEF1A2 and eEF1A1 in onset and progression, respectively, of prostate cancer. These two targets have been studied at DNA and mRNA levels in plasma circulating nucleic acids and in tissues. Materials and Methods The study was approved by the Ethical Committee of the UNITS (n.102, 16.01.20 and n.110 25.01.21). We enrolled a preliminary pool of patients from our hospital with a suspected PCa undergoing a prostate biopsy. Through a blood sample, we tested a specific cfDNA LINE-1, to demonstrate if these repetitive elements can be related to the diagnosis and staging of PCa. The plasma was than prepared and anonymously preserved in a blood bank at our Institute following the method set up by the research group. After the anatomopathological diagnosis of the biopsy, samples of 41 plasma of cancer patients (cases) and 19 plasma of negative cancer patients (controls) were selected for the explorative analysis. The circulating cfDNA/cfRNA were extracted by QIAamp ccfDNA/RNA Kit and stocked into dedicated cryovials in a bank at -80°C. The LINE-1 target in cfDNA were detected by ddPCR technology using Eva Green assays. The cfDNA integrity (cfDI) was obtained from the ratio of longer/shorter fragments following the method set up by the research group. The EEF1A1/A2 DNA and the cDNA of eEF1A1 were evaluated by FAM/HEX probe assays. The EEF1A1/A2 DNA copies, the cDNA of eEF1A1 and cfDI of LINE-1 were compared between cases and controls by statistical evaluation. Results Higher eEF1A1 expression in Gleason 7–8 tissues vs Gleason 4–6 tissues (Gleason ≥ 7, 87% vs. Gleason ≤ 6, 54%; p = 0.033). Patients exhibiting elevated eEF1A1 expression experienced poorer clinical outcomes. To highlight the role of eEF1A1 in aggressive PCa, we used a cell model. In PC-3 hormone-independent cancer cells, but not in non-tumorigenic PZHPV-7 cells, GT75 led to reduced cell viability, increased autophagy, and enhanced cell detachment. Remarkably, in PC-3 cells, GT75 primarily co-localized with the fraction of eEF1A1 bound to actin. Elevated eEF1A1 protein levels could serve as an indicator of aggressive prostate cancer forms. Moreover, targeting eEF1A1 with GT75 impaired cell viability specifically in PC-3 cancer cells but had no effect on PZHPV-7 non- tumorigenic cells, suggesting a distinct role for this protein in cancer cell survival. Preliminary analysis of plasma liquid biopsy data revealed no significant differences between cases and controls in the EEF1A1/EEF1A2 DNA copy ratio. The presence of mRNA from eEF1A1 was the only evaluable variable and no difference was found between cases and controls. In contrast, the cfDI of LINE -1 showed a significant difference between cases and controls, with the cfDI of LINE -1 being lower in cases than in controls (Mann-Whitney test p=0.004). The amount of cfDNA measured by EEF1A1 or EEF1A2 was also higher in cases than controls (Mann-Whitney test p=0.04). The cfDI of LIN the cfDI of LINE -1 in plasma can distinguish cases of prostate cancer from healthy controls.