Biomarkers and liquid biopsy on cfDNA for precision cancer medicine

The research program focuses on the study of biomarkers useful for diagnosis, prognosis and monitoring of the therapeutic responses of cancer patients, for the early detection of tumours and their relapses.

Part of the interest is on the study of the two isoforms of the eukaryotic Elongation Factors A1 and A2 (eEF1A1 and eEF1A2) and their roles in the oncogenesis and progression of human tumors, particularly in breast and prostate cancers. These two proteins are implicated in the oncogenesis and progression of many human tumors, but their role as biomarkers remains to be clarified.

More recently, the interest has been focused on the analysis of free circulating nucleic acids with liquid biopsy for solid tumors, such as breast and prostate cancers, which are considered among the big killers of gender. Liquid biopsy has been the subject of numerous scientific researches that have proven its great usefulness. It has established itself as a non-invasive, repeatable, reliable procedure and a source of information on tumor behaviors. Liquid biopsy consists of a simple draw of peripheral blood or other biological fluids, including urine. Circulating molecules such as nucleic acids or circulating cancer cells and exosomes, which are sources of valuable information about the tumor, can be analyzed from these fluids.

Many studies have shown that liquid biopsy analyses on circulating nucleic acids (DNA and RNA) can give information that are representative of the tumor heterogeneity.  In addition, liquid biopsy on circulating cell-free nucleic acids has been proved to anticipate drug-resistance and the recurrence of the disease.

Cell-free circulating DNA (cfDNA) is the fragmented DNA released by cells in apoptosis or necrosis. CfDNA can be found, in addition to blood, in other body fluids such as urine, cerebrospinal fluid, pleural fluid and saliva. It is possible to correlate tumors to the quantification of the total cfDNA or  to analyze the tumor cfDNA (ctDNA) going to search for mutations or for quantification of the copy number of target genes to obtain information on outcomes and response to therapies.

ONGOING PROJECTS

1.Expression of eEF1A1/A2 in human cancer tissues and their role in tumours.

The research team works on the study of eEF1A1 and eEF1A2 expression in human cancer tissues and the evaluation of eEF1A1, eEF1A2 in liquid biopsy. It is investigating the role of eEF1A1 and eEF1A2 in breast and prostate cancers using a system developed by the group for immunoistochemistry and immunofluorescence analyses. In addition, the group is analyzing the expression of the two genes in tissues with the PCR digital droplet system (ddPCR).

2. CfDNA and cfDI analysis in patients with breast cancer onset.

Breast cancer accounts for 30% of total female cancers and, in Italy, it has an estimate of at least 53,000 new cases per year. In women, breast cancer accounts for 28% of causes of cancer death before the age of 50, 20% between the age of 50 and 69 and 14% after the age of 70.

In liquid biopsy, the group is studying the integrity of free circulating DNA (cfDI) in the plasma of patients with primary breast cancer onset to assess its predictivity value for the onset of the disease and its severity.

From https://doi.org/10.1016/j.bdq.2019.100087

Basically, cfDI is the ratio for a nested target on the cfDNA, of the large fragment’s quantity over the short one. CfDI has been shown to change significantly in the presence of a tumor and when recurrence and progression of the tumor is occured. The research team is analysing the cfDI of traits of ALU and LINE-1 sequences. ALU and LINE-1 are part of the non-Long Terminal Repeat retrotransposons sequences. Transposable elements play an essential role in maintaining genomic integrity, and their deregulation can cause genome instability, implicated in the tumor development. LINE-1s belong to the LINEs class (Long Interspersed Nuclear Elements) and account for about 17% of the human genome. LINE-1s encode for proteins needed for their own amplification, which is why autonomous elements are defined. ALU elements belong to the SINEs class (Short Interpersed Elements), are primates-specific elements and account for 11% of the genomic sequence.

The research team obtained the consent of the C.E.U.R. FVG for liquid biopsy on patients and controls. They are analyzing the cfDI of ALU and LINE-1 in the plasma of patients diagnosed with primary breast cancer onset and in healthy control subjects. The study involves the use of ddPCR for which the group has developed the cfDI analyses on ALU and LINE-1. In addition, the group's interest is to assess the amount of circulating cfDNA with gene-target system in ddPCR using EEF1A1 and EEF1A2 as reference genes.