Phd in MOLECULAR MEDICINE

Thesis title: Innovative approaches in liquid biopsy towards a more accurate precision medicine

As one of the milestones in the history of cancer research, liquid biopsies (LB) have crucially impacted the world of clinical oncology. Although solid literature corroborates the validity of LB methodologies, only few have been approved in the clinic. Moreover, as many efforts have been spent to refine technologies for the isolation and downstream analyses of liquid biomarkers, critical limitations remain. Under these premises, the aim of this work has been to embrace several challenges in the field of LB, improving the clinical relevance of LB assays and ultimately looking forward to new applications. Structurally, three major challenges (clinical, technical and innovation) have been addressed, focusing on two major liquid analytes: circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs). Despite the consistent bulk of evidence supporting the clinical scope of LB, its translation into the clinical practice is still limited to date. In this context, the importance of ctDNA in monitoring cancer features at treatment failure has been particularly stressed. In patients with advanced non-small cell lung cancer, the analyses conducted on ctDNA showed that patients initially harboring a KRAS G12C mutation in tumor tissue can switch to a wild-type (wt) RAS in plasma and vice versa. Similarly, in RAS mutant metastatic colorectal cancer (mCRC) patients, it has been observed a negative selection of mutant RAS clones, significantly associated to the use of bevacizumab as first-line treatment. Further investigation of the genomic features related to RAS mutation clearance resulted in the identification of a group of patients defined “neo-RAS wild-type”. RAS clearance has been found to be indicative of improved overall survival and post progression survival compared to patients who maintained RAS mutations. Therefore, serial ctDNA evaluations represent a precious tool for a real-time identification of variations in the molecular profile of the tumor along with treatment failure, not only helping clinicians to better match patients to interventional biomarker-targeted therapies, but also predicting clinical outcome. Besides tumor monitoring, the detection and analysis of ctDNA for the early diagnosis of cancer clearly represent one of the most interesting and promising clinical applications of LB, although still limited. In collaboration with the thoracic surgery Unit of Policlinico Umberto I, a study exploring the presence and the molecular features of plasmatic ctDNA samples from patients with evidenced ground glass opacity is currently ongoing. Preliminary data show that our workflow enables ctDNA isolation and analysis, despite an optimization in terms of quality and quantity of the extracted ctDNA is required. With regards to the technological challenges, a crucial limit in LB is the low amount of analyte available for the investigations. Thus, the sensitivity and specificity of LB assays need to be extremely high to avoid false positive and false negative results. In our experience one of the most important methodological problems has been to determine whether the absence of a specific mutation is related to the lack of that mutation or to the absence of ctDNA. For this reason, to confirm the dynamics of RAS mutation clearance in the plasma of mCRC patients, methylation and sequencing analyses have been conducted. A further technical limitation in the field of LB is the concomitant release of CTCs as either single cells or clusters in the bloodstream. Despite their similarities, these two analytes bring different types of information about cancer. The establishment of a new pipeline for the isolation and subsequent analysis of both CTCs and CTC clusters (CTCCL) from a single blood sample produced successful results that clearly constitute an important advance in the analysis of these biomarkers. During the last years, particular attention has been paid to the use of circulating tumor RNA as a liquid biomarker. Providing different and complementary information about cancer compared to other LB analytes, the analysis of circulating RNAs hold promise of great clinical potential, although technically challenging. With the purpose of investigate the impact of first-line chemotherapy plus immunotherapy in patients with advanced non-small cell lung cancer using both ctDNA and ctRNA, a protocol for the isolation and sequencing of circulating tumor RNA has been designed. However, further optimizations are needed, as the low concentration of extracted RNA was not sufficient to perform sequencing analyses. Lastly, in the context of CTCs, a major challenge is represented by the fact that, although the prognostic value of CTCs counts is widely recognized, no further applications are currently approved. Since a single enumeration of CTC at baseline being generally used to predict patients’ outcomes, investigating the prognostic and predictive value of serial CTCs counts from 218 mCRC patients offered the opportunity to make a significant progress in the quantitative analysis of CTCs, overcoming the concept of a single count. This retrospective study unearthed innovative potential use of CTCs trajectories in mCRC patients during first-line treatments, which were found to be more informative and also accurate compared to a single CTC count. In conclusion, despite numerous challenges remain in this field, this work aimed to contribute to the resolution of previous LB limitations and underline the importance of this tool in precision medicine.