Liquid Biopsy in the Management of Breast Cancer Patients: Where Are We Now and Where Are We Going.

Liquid biopsy (LB) is an emerging diagnostic tool that analyzes biomarkers in the blood (and possibly in other body fluids) to provide information about tumor genetics and response to therapy. This review article provides an overview of LB applications in human cancer with a focus on breast cancer patients. LB methods include circulating tumor cells and cell-free tumor products, such as circulating tumor DNA. LB has shown potential in detecting cancer at an early stage, monitoring tumor progression and recurrence, and predicting patient response to therapy. Several studies have demonstrated its clinical utility in breast cancer patients. However, there are limitations to LB, including the lack of standardized assays and the need for further validation. Future potential applications of LB include identifying the minimal residual disease, early detection of recurrence, and monitoring treatment response in various cancer types. LB represents a promising non-invasive diagnostic tool with potential applications in breast cancer diagnosis, treatment, and management. Further research is necessary to fully understand its clinical utility and overcome its current limitations.

Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling.

Molecular characterization of Circulating Tumor Cells (CTCs) is still challenging, despite attempts to minimize the drawbacks of Whole Genome Amplification (WGA). In this paper, we propose a Next-Generation Sequencing (NGS) optimized protocol based on molecular tagging technology, in order to detect CTCs mutations while skipping the WGA step. MDA-MB-231 and MCF-7 cell lines, as well as leukocytes, were sorted into pools (2-5 cells) using a DEPArray™ system and were employed to set up the overall NGS procedure. A substantial reduction of reagent volume for the preparation of libraries was performed, in order to fit the limited DNA templates directly derived from cell lysates. Known variants in TP53, KRAS, and PIK3CA genes were detected in almost all the cell line pools (35/37 pools, 94.6%). No additional alterations, other than those which were expected, were found in all tested pools and no mutations were detected in leukocytes. The translational value of the optimized NGS workflow is confirmed by sequencing CTCs pools isolated from eight breast cancer patients and through the successful detection of variants. In conclusion, this study shows that the proposed NGS molecular tagging approach is technically feasible and, compared to traditional NGS approaches, has the advantage of filtering out the artifacts generated during library amplification, allowing for the reliable detection of mutations and, thus, making it highly promising for clinical use.

Autophagy induced by SAHA affects mutant P53 degradation and cancer cell survival.

Missense mutations in the TP53 gene produce mutant p53 (mutp53) proteins which may acquire oncogenic properties favoring chemoresistance, cell migration, and metastasis. The exploitation of cellular pathways that promote mutp53 degradation may reduce cell proliferation and invasion as well as increase the sensitivity to anticancer drugs, with a strong impact on current cancer therapies. In the last years, several molecules have been characterized for their ability to induce the degradation of mutp53 through the activation of autophagy. Here, we investigated the correlation between autophagy and mutp53 degradation induced by suberoylanilide hydroxamic acid (SAHA), an FDA-approved histone deacetylase inhibitor. In the human cancer lines MDA-MB-231 (mutp53-R280K) and DLD1 (mutp53-S241F), SAHA induced a significant mutp53 degradation. However, such degradation correlated with autophagy induction only in MDA-MB-231 cells, being counteracted by autophagy inhibition, which also increased SAHA-induced cell death. Conversely, in DLD1 cells SAHA triggered a low level of autophagy despite promoting a strong decrease in mutp53 level, and autophagy inhibition did not change either mutp53 levels or sensitivity to this drug. We conclude that autophagy can be a relevant pathway for mutp53 degradation induced by SAHA, but its contribution to mutp53 destabilization and the consequences on cell death are likely context-dependent.